Advances in DNA Sequencing Technologies

Advances in DNA Sequencing Technologies Abstract Recent advances in DNA sequencing technologies have led to efficient methods for determining the sequence of DNA. DNA sequencing was born in 1977 when Sanger et al proposed the chain termination method and Maxam and Gilbert proposed their own method in the same year. Sangers method was proven to be the most favourable out of the two. Since the birth of DNA sequencing, efficient DNA sequencing technologies was being produced, as Sangers method was laborious, time consuming and expensive; Hood et al proposed automated sequencers involving dye-labelled terminators. Due to the lack of available computational power prior to 1995, sequencing an entire bacterial genome was considered out of reach. This became a reality when Venter and Smith proposed shotgun sequencing in 1995. Pyrosequencing was introduced by Ronagi in 1996 and this method produce the sequence in real-time and is applied by 454 Life Sciences. An indirect method of sequencing DNA was proposed by Drmanac in 1987 called sequen cing by hybridisation and this method lead to the DNA array used by Affymetrix. Nanopore sequencing is a single-molecule sequencing technique and involves single-stranded DNA passing through lipid bilayer via an ion channel, and the ion conductance is measured. Synthetic Nanopores are being produced in order to substitute the lipid bilayer. Illumina sequencing is one of the latest sequencing technologies to be developed involving DNA clustering on flow cells and four dye-labelled terminators performing reverse termination. DNA sequencing has not only been applied to sequence DNA but applied to the real world. DNA sequencing has been involved in the Human genome project and DNA fingerprinting. Introduction Reliable DNA sequencing became a reality in 1977 when Frederick Sanger who perfected the chain termination method to sequence the genome of bacteriophage ?X174 [1][2]. Before Sangers proposal of the chain termination method, there was the plus and minus method, also presented by Sanger along with Coulson [2]. The plus and minus method depended on the use of DNA polymerase in transcribing the specific sequence DNA under controlled conditions. This method was considered efficient and simple, however it was not accurate [2]. As well as the proposal of the chain termination sequencing by Sanger, another method of DNA sequencing was introduced by Maxam and Gilbert involving restriction enzymes, which was also reported in 1977, the same year as Sangers method. The Maxamm and Gilbert method shall be discussed in more detail later on in this essay. Since the proposal of these two methods, spurred many DNA sequencing methods and as the technology developed, so did DNA sequencing. In this lite rature review, the various DNA sequencing technologies shall be looked into as well their applications in the real world and the tools that have aided sequencing DNA e.g. PCR. This review shall begin with the discussion of the chain termination method by Sanger. The Chain Termination Method Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. In order to remove the 3-hydroxyl group and replace it with a proton, the triphosphate has to undergo a chemical procedure [1]. There is a different procedure employed for each of the triphosphate groups. Preparation of ddATP was produced from the starting material of 3-O-tosyl-2-deoxyadenosine which was treated with sodium methoxide in dimethylformamide to produce 2,3-dideoxy-2,3-didehydroadenosine, which is an unsaturated compound [4]. The double bond between carbon 2 and 3 of the cyclic ether was then hydrogenated with a palladium-on-carbon catalyst to give 2,3-dideoxyadenosine (ddA). The ddA (ddA) was then phosphorylated in order add the triphosphate group. Purification then took place on DEAE-Sephadex column using a gradient of triethylamine carbonate at pH 8.4. Figure 2 is schematic representation to produce ddA prior to phosphorylation. In the preparation of ddTTP (Figure 3), thymidine was tritylated (+C(Ph3)) at the 5-position and a methanesulphonyl (+CH3SO2) group was introduced at the 3-OH group[5]. The methanesulphonyl group was substituted with iodine by refluxing the compound in 1,2-dimethoxythane in the presence of NaI. After chromatography on a silica column the 5-trityl-3-iodothymidine was hydrogenated in 80% acetic acid to remove the trityl group. The resultant 3-iodothymidine was hydrogenated to produce 23-dideoxythymidine which subsequently was phosphorylated. Once phosphorylated, ddTTP was then purified on a DEAE-sephadex column with triethylammonium-hydrogen carbonate gradient. Figure 3 is a schematic representation to produce ddT prior phosphorylation. When preparing ddGTP, the starting material was N-isobutyryl-5-O-monomethoxytrityldepxyguanosine [1]. After the tosylation of the 3-OH group the compound was then converted to the 23-didehydro derivative with sodium methoxide. Then the isobutyryl group was partly removed during this treatment of sodium methoxide and was removed completely by incubation in the presence of NH3 overnight at 45oC. During the overnight incubation period, the didehydro derivative was reduced to the dideoxy derivative and then converted to the triphosphate. The triphosphate was purified by the fractionation on a DEAE-Sephadex column using a triethylamine carbonate gradient. Figure 4 is a schematic representation to produce ddG prior phosphorylation. Preparing the ddCTP was similar to ddGTP, but was prepared from N-anisoyl-5-O-monomethoxytrityldeoxycytidine. However the purification process was omitted for ddCTP, as it produced a very low yield, therefore the solution was used directly in the experiment described in the paper [2]. Figure 5 is a schematic representation to produce ddC prior phosphorylation. With the four dideoxy samples now prepared, the sequencing procedure can now commence. The dideoxy samples are in separate tubes, along with restriction enzymes obtained from ?X174 replicative form and the four dNTPs [2]. The restriction enzymes and the dNTPs begin strand synthesis and the ddNTP is incorporated to the growing polynucleotide and terminates further strand synthesis. This is due to the lack of the hydroxyl group at the 3 position of ddNTP which prevents the next nucleotide to attach onto the strand. The four tubes are separate by gel-electrophoresis on acrylamide gels (see Gel-Electrophoresis). Figure 6 shows the sequencing procedure. Reading the sequence is straightforward [1]. The first band that moved the furthest is located, this represents the smallest piece of DNA and is the strand terminated by incorporation of the dideoxynucleotide at the first position in the template. The track in which this band occurs is noted. For example (shown in Figure 6), the band that moved the furthest is in track A, so the first nucleotide in the sequence is A. To find out what the next nucleotide, the next most mobile band corresponding to DNA molecule which is one nucleotide longer than the first, and in this example, the band is on track T. Therefore the second nucleotide is T, and the overall sequence so far is AT. The processed is carried on along the autoradiograph until the individual bands start to close in and become inseparable, therefore becoming hard to read. In general it is possible to read upto 400 nucleotides from one autoradiograph with this method. Figure 7 is a schematic representation of an autoradiograph. E ver since Sanger perfected the method of DNA sequencing, there have been advances methods of sequencing along with the achievements. Certain achievements such as the Human genome project and shall be discussed later on in this review. Gel-Electrophoresis Gel-Electrophoresis is defined as the movement of charged molecules in an electric field [1][8]. DNA molecules, like many other biological compounds carry an electric charge. With the case of DNA, this charge is negative. Therefore when DNA is placed in an electric field, they migrate towards the positive pole (as shown in figure 8). There are three factors which affect the rate of migration, which are shape, electrical charge and size. The polyacrylamide gel comprises a complex network of pores through which the molecules must travel to reach the anode. Maxam and Gilbert Method The Maxam and Gilbert method was proposed before Sanger Method in the same year. While the Sangers method involves enzymatic radiolabelled fragments from unlabelled DNA strands [2]. The Maxam-Gilbert method involves chemical cleavage of prelabelled DNA strands in four different ways to form the four different collections of labelled fragments [6][7]. Both methods use gel-electrophoresis to separate the DNA target molecules [8]. However Sangers Chain Termination method has been proven to be simpler and easier to use than the Maxam and Gilbert method [9]. As a matter of fact, looking through the literature text books, Sangers method of DNA sequencing have been explained rather than Maxam and Gilberts [1][3][9][10]. With Maxam and Gilberts method there are two chemical cleavage reactions that take place [6][7]. One of the chemical reaction take places with guanine and the adenine, which are the two purines and the other cleaves the DNA at the cytosine and thymin e, the pyrimidines. For the cleavage reaction, specific reagents are used for each of the reaction. The purine specific reagent is dimethyl sulphate and the pyrimidine specific reagent is hydrazine. Each of these reactions are done in a different way, as each of the four bases have different chemical properties. The cleavage reaction for the guanine/adenine involves using dimethyl sulphate to add a methyl group to the guanines at the N7 position and at the N3 position at the adenines [7]. The glycosidic bond of a methylated adenines is unstable and breaks easily on heating at neutral pH, leaving the sugar free. Treatment with 0.1M alkali at 90oC then will cleave the sugar from the neighbouring phosphate groups. When the resulting end-labelled fragments are resolved on a polyacrylamide gel, the autoradiograph contains a pattern a dark and light bands. The dark bands arise from the breakage at the guanines, which methylate at a rate which is 5-fold faster than adenines. From this reac tion the guanine appear stronger than the adenosine, this can lead to a misinterpretation. Therefore an Adenine-Enhanced cleavage reaction takes place. Figure 9 shows the structural changes of guanine when undergoing the structural modifications involved in Maxam-Gilbert sequencing. With an Adenine-Enhanced cleavage, the glycosidic bond of methylated adenosine is less stable than that of methylated guanosine, thus gentle treatment with dilute acid at the methylation step releases the adenine, allowing darker bands to appear on the autoradiograph [7]. The chemical cleavage for the cytosine and thymine residues involves hydrazine instead of dimethyl sulphate. The hydrazine cleaves the base and leaving ribosylurea [7]. After partial hydrazinolysis in 15-18M aqueous hydrazine at 20oC, the DNA is cleaved with 0.5M piperidine. The piperidine (a cyclic secondary amine), as the free base, displaces all the products of the hydrazine reaction from the sugars and catalyzses the b-elimination of the phosphates. The final pattern contains bands of the similar intensity from the cleavages at the cytosines and thymines. As for cleavage for the cytosine, the presence of 2M NaCl preferentially suppresses the reaction of thymine with hydrazine. Once the cleavage reaction has taken place each original strand is broken into a labelled fragment and an unlabelled fragment [7]. All the labelled fragments start at the 5 end of the strand and terminate at the base that precedes the site of a nucleotide along the original strand. Only the labelled fragmen ts are recorded on the gel electrophoresis. Dye-labelled terminators For many years DNA sequencing has been done by hand, which is both laborious and expensive[3]. Before automated sequencing, about 4 x 106 bases of DNA had been sequenced after the introduction of the Sangers method and Maxam Gilbert methods [11]. In both methods, four sets of reactions and a subsequent electrophoresis step in adjacent lanes of a high-resolution polyacrylamide gel. With the new automated sequencing procedures, four different fluorophores are used, one in each of the base-specific reactions. The reaction products are combined and co-electrophoresed, and the DNA fragments generated in each reaction are detected near the bottom of the gel and identified by their colour. As for choosing which DNA sequencing method to be used, Sangers Method was chosen. This is because Sangers method has been proven to be the most durable and efficient method of DNA sequencing and was the choice of most investigators in large scale sequencing [12]. Figure 10 shows a typical sequence is ge nerated using an automated sequencer. The selection of the dyes was the central development of automated DNA sequencing [11]. The fluorophores that were selected, had to meet several criteria. For instance the absorption and emission maxima had to be in the visible region of the spectrum [11] which is between 380 nm and 780 nm [10], each dye had to be easily distinguishable from one another [11]. Also the dyes should not impair the hybridisation of the oligonucleotide primer, as this would decrease the reliability of synthesis in the sequencing reactions. Figure 11 shows the structures of the dyes which are used in a typical automated sequencing procedure, where X is the moiety where the dye will be bound to. Table 1 shows which dye is covalently attached to which nucleotide in a typical automated DNA sequencing procedure Dye Nucleotide Attached Flourescein Adenosine NBD Thymine Tetramethylrhodamine Guanine Texas Red Cytosine In designing the instrumentation of the florescence detection apparatus, the primary consideration was sensitivity. As the concentration of each band on the co-electrophoresis gel is around 10 M, the instrument needs to be capable of detecting dye concentration of that order. This level of detection can readily be achieved by commercial spectrofluorimeter systems. Unfortunately detection from a gel leads to a much higher background scatter which in turn leads to a decrease in sensitivity. This is solved by using a laser excitation source in order to obtain maximum sensitivity [11]. Figure 12 is schematic diagram of the instrument with the explanation of the instrumentation employed. When analyzing data, Hood had found some complications [11]. Firstly the emission spectra of the different dyes overlapped, in order to overcome this, multicomponent analysis was employed to determine the different amounts of the four dyes present in the gel at any given time. Secondly, the different dye molecules impart non-identical electrophoretic mobilities to the DNA fragments. This meant that the oligonucleotides were not equal base lengths. The third major complication was in analyzing the data comes from the imperfections of the enzymatic methods, for instance there are often regions of the autoradiograph that are difficult to sequence. These complications were overcome in five steps [11] High frequency noise is removed by using a low-pass Fourier filter. A time delay (1.5-4.5 s) between measurements at different wavelength is partially corrected for by linear interpolation between successive measurements. A multicomponent analysis is performed on each set of four data points; this computation yields the amount of each of the four dyes present in the detector as a function of time. The peaks present in the data are located The mobility shift introduced by the dyes is corrected for using empirical determined correction factors. Since the publication of Hoods proposal of the fluorescence detection in automated DNA sequence analysis. Research has been made on focussed on developing which are better in terms of sensitivity [12]. Bacterial and Viral Genome Sequencing (Shotgun Sequencing) Prior to 1995, many viral genomes have been sequenced using Sangers chain termination technique [13], but no bacterial genome has been sequenced. The viral genomes that been sequenced are the 229 kb genome of cytomegalovirus [14], and the 192 kb genome of vaccinia [15], the 187 kb mitochondrial and 121 kb cholorophast genomes of Marchantia polymorpha have been sequenced [16]. Viral genome sequencing has been based upon the sequencing of clones usually derived from extensively mapped restriction fragments, or ? or cosmid clones [17]. Despite advances in DNA sequencing technology, the sequencing of genomes has not progressed beyond clones on the order of the size of the ~ 250kb, which is due to the lack of computational approaches that would enable the efficient assembly of a large number of fragments into an ordered single assembly [13][17]. Upon this, Venter and Smith in 1995 proposed Shotgun Sequencing and enabled Haemophilus influenzae (H. influenzae) to become the first bacterial genome to be sequenced [13][17]. H. influenzae was chosen as it has a similar base composition as a human does with 38 % of sequence made of G + C. Table 2 shows the procedure of the Shotgun Sequencing [17]. When constructing the library ultrasonic waves were used to randomly fragment the genomic DNA into fairly small pieces of about the size of a gene [13]. The fragments were purified and then attached to plasmid vectors[13][17]. The plasmid vectors were then inserted into an E. coli host cell to produce a library of plasmid clones. The E. coli host cell strains had no restriction enzymes which prevented any deletions, rearrangements and loss of the clones [17]. The fragments are randomly sequenced using automated sequencers (Dye-Labelled terminators), with the use of T7 and SP6 primers to sequence the ends of the inserts to enable the coverage of fragments by a factor of 6 [17]. Table 2 (Reference 17) Stage Description Random small insert and large insert library construction Shear genomic DNA randomly to ~2 kb and 15 to 20 kb respectively Library plating Verify random nature of library and maximize random selection of small insert and large insert clones for template production High-throughput DNA sequencing Sequence sufficient number of sequences fragments from both ends for 6x coverage Assembly Assemble random sequence fragments and identity repeat regions Gap Closure Physical gaps Order all contigs (fingerprints, peptide links, ÃŽ », clones, PCR) and provide templates for closure Sequence gaps Complete the genome sequence by primer walking Editing Inspect the sequence visually and resolve sequence ambiguities, including frameshifts Annotation Identify and describe all predicted coding regions (putative identifications, starts and stops, role assignments, operons, regulatory regions) Once the sequencing reaction has been completed, the fragments need to be assembled, and this process is done by using the software TIGR Assembler (The Institute of Genomic Research) [17]. The TIGR Assembler simultaneously clusters and assembles fragments of the genome. In order to obtain the speed necessary to assemble more than 104 fragments [17], an algorithm is used to build up the table of all 10-bp oligonucleotide subsequences to generate a list of potential sequence fragment overlaps. The algorithm begins with the initial contig (single fragment); to extend the contig, a candidate fragment is based on the overlap oligonucleotide content. The initial contig and candidate fragment are aligned by a modified version of the Smith-Waterman [18] algorithm, which allows optional gapped alignments. The contig is extended by the fragment only if strict criteria of overlap content match. The algorithm automatically lowers these criteria in regions of minimal coverage and raises them in r egions with a possible repetitive element [17]. TIGR assembler is designed to take advantage of huge clone sizes [17]. It also enforces a constraint that sequence from two ends of the same template point toward one another in the contig and are located within a certain range of the base pair [17]. Therefore the TIGR assembler provides the computational power to assemble the fragments. Once the fragments have been aligned, the TIGR Editor is used to proofread the sequence and check for any ambiguities in the data [17]. With this technique it does required precautionary care, for instance the small insert in the library should be constructed and end-sequenced concurrently [17]. It is essential that the sequence fragments are of the highest quality and should be rigorously check for any contamination [17]. Pyrosequencing Most of the DNA sequencing required gel-electrophoresis, however in 1996 at the Royal Institute of Technology, Stockholm, Ronaghi proposed Pyrosequencing [19][20]. This is an example of sequencing-by-synthesis, where DNA molecules are clonally amplified on a template, and this template then goes under sequencing [25]. This approach relies on the detection of DNA polymerase activity by enzymatic luminometric inorganic pyrophosphate (PPi) that is released during DNA synthesis and goes under detection assay and offers the advantage of real-time detection [19]. Ronaghi used Nyren [21] description of an enzymatic system consisting of DNA polymerase, ATP sulphurylase and lucifinerase to couple the release of PPi obtained when a nucleotide is incorporated by the polymerase with light emission that can be easily detected by a luminometer or photodiode [20]. When PPi is released, it is immediately converted to adenosine triphosphate (ATP) by ATP sulphurylase, and the level of generated ATP is sensed by luciferase-producing photons [19][20][21]. The unused ATP and deoxynucleotide are degraded by the enzyme apyrase. The presence or absence of PPi, and therefore the incorporation or nonincorporation of each nucleotide added, is ultimately assessed on the basis of whether or not the photons are detected. There is minimal time lapse between these events, and the conditions of the reaction are such that iterative addition of the nucleotides and PPi detection are possible. The release of PPi via the nucleotide incorporation, it is detected by ELIDA (Enzymatic Luminometric Inorganic pyrophosphate Detection Assay) [19][21]. It is within the ELIDA, the PPi is converted to ATP, with the help of ATP sulfurylase and the ATP reacts with the luciferin to generate the light at more than 6 x 109 photons at a wavelength of 560 nm which can be detected by a photodiode, photomultiplier tube, or charge-coupled device (CCD) camera [19][20]. As mentioned before, the DNA molecules need to be amplified by polymerase chain reaction (PCR which is discussed later Ronaghi observed that dATP interfered with the detection system [19]. This interference is a major problem when the method is used to detect a single-base incorporation event. This problem was rectified by replacing the dATP with dATPaS (deoxyadenosine a–thiotrisulphate). It is noticed that adding a small amount of the dATP (0.1 nmol) induces an instantaneous increase in the light emission followed by a slow decrease until it reached a steady-state level (as Figure 11 shows). This makes it impossible to start a sequencing reaction by adding dATP; the reaction must instead be started by addition of DNA polymerase. The signal-to-noise ratio also became higher for dATP compared to the other nucleotides. On the other hand, addition of 8 nmol dATPaS (80-fold higher than the amount of dATP) had only a minor effect on luciferase (as Figure 14 shows). However dATPaS is less than 0.05% as effective as dATP as a substrate for luciferase [19]. Pyrosequencing is adapted by 454 Life Sciences for sequencing by synthesis [22] and is known as the Genome Sequencer (GS) FLX [23][24]. The 454 system consist of random ssDNA (single-stranded) fragments, and each random fragment is bound to the bead under conditions that allow only one fragment to a bead [22]. Once the fragment is attached to the bead, clonal amplification occurs via emulsion. The emulsified beads are purified and placed in microfabricated picolitre wells and then goes under pyrosequencing. A lens array in the detection of the instrument focuses luminescene from each well onto the chip of a CCD camera. The CCD camera images the plate every second in order to detect progression of the pyrosequencing [20][22]. The pyrosequencing machine generates raw data in real time in form of bioluminescence generated from the reactions, and data is presented on a pyrogram [20] Sequencing by Hybridisation As discussed earlier with chain-termination, Maxamm and Gilbert and pyrosequencing, these are all direct methods of sequencing DNA, where each base position is determined individually [26]. There are also indirect methods of sequencing DNA in which the DNA sequence is assembled based on experimental determination of oligonucleotide content of the chain. One promising method of indirect DNA sequencing is called Sequencing by Hybridisation in which sets of oligonucleotide probes are hybridised under conditions that allow the detection of complementary sequences in the target nucleic acid [26]. Sequencing by Hybridisation (SBH) was proposed by Drmanac et al in 1987 [27] and is based on Dotys observation that when DNA is heated in solution, the double-strand melts to form single stranded chains, which then re-nature spontaneously when the solution is cooled [28]. This results the possibility of one piece of DNA recognize another. And hence lead to Drmanac proposal of oligonucleotides pro bes being hybridised under these conditions allowing the complementary sequence in the DNA target to be detected [26][27]. In SBH, an oligonucleotide probe (n-mer probe where n is the length of the probe) is a substring of a DNA sample. This process is similar to doing a keyword search in a page full of text [29]. The set of positively expressed probes is known as the spectrum of DNA sample. For example, the single strand DNA 5GGTCTCG 3 will be sequenced using 4-mer probes and 5 probes will hybridise onto the sequence successfully. The remaining probes will form hybrids with a mismatch at the end base and will be denatured during selective washing. The five probes that are of good match at the end base will result in fully matched hybrids, which will be retained and detected. Each positively expressed serves as a platform to decipher the next base as is seen in Figure 16. For the probes that have successfully hybridised onto the sequence need to be detected. This is achieved by labelling the probes with dyes such as Cyanine3 (Cy3) and Cyanine5 (Cy5) so that the degree of hybridisation can be detected by imaging devices [29]. SBH methods are ideally suited to microarray technology due to their inherent potential for parallel sample processing [29]. An important advantage of using of using a DNA array rather than a multiple probe array is that all the resulting probe-DNA hybrids in any single probe hybridisation are of identical sequence [29]. One of main type of DNA hybridisation array formats is oligonucleotide array which is currently patented by Affymetrix [30]. The commercial uses of this shall be discussed under application of the DNA Array (Affymetrix). Due to the small size of the hybridisation array and the small amount of the target present, it is a challenge to acquire the signals from a DNA Array [29]. These signals must first be amplified b efore they can be detected by the imaging devices. Signals can be boosted by the two means; namely target amplification and signal amplification. In target amplification such as PCR, the amount of target is increased to enhance signal strength while in signal amplification; the amount of signal per unit is increased. Nanopore Sequencing Nanopore sequencing was proposed in 1996 by Branton et al, and shows that individual polynucleotide molecules can be characterised using a membrane channel [31]. Nanopore sequencing is an example of single-molecule sequencing, in which the concept of sequencing-by-synthesis is followed, but without the prior amplification step [24]. This is achieved by the measurement of ionic conductance of a nucleotide passing through a single ion channels in biological membranes or planar lipid bilayer. The measurement of ionic conductance is routine neurobiology and biophysics [31], as well as pharmacology (Ca+ and K+ channel)[32] and biochemistry[9]. Most channels undergo voltage-dependant or ligand dependant gating, there are several large ion channels (i.e. Staphylococcus aureus a-hemolysin) which can remain open extended periods, thereby allowing continuous ionic current to flow across a lipid bilayer [31]. If a transmembrane voltage applied across an open channel of appropriate size should d raw DNA molecules through the channel as extended linear chains whose presence would detect reduce ionic flow. It was assumed, that the reduction in the ionic flow would lead to single channel recordings to characterise the length and hence lead to other characteristics of the polynucleotide. In the proposal by Branton, a-hemolysin was used to form a single channel across a lipid bilayer separating two buffer-filled compartment [31]. a-Hemolysin is a monomeric, 33kD, 293 residue protein that is secreted by the human pathogen Staphylococcus aureus [33]. The nanopore are produced when a-hemolysin subsunits are introduced into a buffered solution that separates lipid bilayer into two compartments (known as cis and trans): the head of t Advances in DNA Sequencing Technologies Advances in DNA Sequencing Technologies Abstract Recent advances in DNA sequencing technologies have led to efficient methods for determining the sequence of DNA. DNA sequencing was born in 1977 when Sanger et al proposed the chain termination method and Maxam and Gilbert proposed their own method in the same year. Sangers method was proven to be the most favourable out of the two. Since the birth of DNA sequencing, efficient DNA sequencing technologies was being produced, as Sangers method was laborious, time consuming and expensive; Hood et al proposed automated sequencers involving dye-labelled terminators. Due to the lack of available computational power prior to 1995, sequencing an entire bacterial genome was considered out of reach. This became a reality when Venter and Smith proposed shotgun sequencing in 1995. Pyrosequencing was introduced by Ronagi in 1996 and this method produce the sequence in real-time and is applied by 454 Life Sciences. An indirect method of sequencing DNA was proposed by Drmanac in 1987 called sequen cing by hybridisation and this method lead to the DNA array used by Affymetrix. Nanopore sequencing is a single-molecule sequencing technique and involves single-stranded DNA passing through lipid bilayer via an ion channel, and the ion conductance is measured. Synthetic Nanopores are being produced in order to substitute the lipid bilayer. Illumina sequencing is one of the latest sequencing technologies to be developed involving DNA clustering on flow cells and four dye-labelled terminators performing reverse termination. DNA sequencing has not only been applied to sequence DNA but applied to the real world. DNA sequencing has been involved in the Human genome project and DNA fingerprinting. Introduction Reliable DNA sequencing became a reality in 1977 when Frederick Sanger who perfected the chain termination method to sequence the genome of bacteriophage ?X174 [1][2]. Before Sangers proposal of the chain termination method, there was the plus and minus method, also presented by Sanger along with Coulson [2]. The plus and minus method depended on the use of DNA polymerase in transcribing the specific sequence DNA under controlled conditions. This method was considered efficient and simple, however it was not accurate [2]. As well as the proposal of the chain termination sequencing by Sanger, another method of DNA sequencing was introduced by Maxam and Gilbert involving restriction enzymes, which was also reported in 1977, the same year as Sangers method. The Maxamm and Gilbert method shall be discussed in more detail later on in this essay. Since the proposal of these two methods, spurred many DNA sequencing methods and as the technology developed, so did DNA sequencing. In this lite rature review, the various DNA sequencing technologies shall be looked into as well their applications in the real world and the tools that have aided sequencing DNA e.g. PCR. This review shall begin with the discussion of the chain termination method by Sanger. The Chain Termination Method Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. Sanger discovered that the inhibitory activity of 23-didoxythymidine triphosphate (ddTTP) on the DNA polymerase I was dependent on its incorporation with the growing oligonucleotide chain in the place of thymidylic acid (dT) [2]. In the structure of ddT, there is no 3-hydroxyl group, by there is a hydrogen group in place. With the hydrogen in place of the hydroxyl group, the chain cannot be extended any further, so a termination occurs at the position where dT is positioned. Figure 1 shows the structure of dNTP and ddNTP. In order to remove the 3-hydroxyl group and replace it with a proton, the triphosphate has to undergo a chemical procedure [1]. There is a different procedure employed for each of the triphosphate groups. Preparation of ddATP was produced from the starting material of 3-O-tosyl-2-deoxyadenosine which was treated with sodium methoxide in dimethylformamide to produce 2,3-dideoxy-2,3-didehydroadenosine, which is an unsaturated compound [4]. The double bond between carbon 2 and 3 of the cyclic ether was then hydrogenated with a palladium-on-carbon catalyst to give 2,3-dideoxyadenosine (ddA). The ddA (ddA) was then phosphorylated in order add the triphosphate group. Purification then took place on DEAE-Sephadex column using a gradient of triethylamine carbonate at pH 8.4. Figure 2 is schematic representation to produce ddA prior to phosphorylation. In the preparation of ddTTP (Figure 3), thymidine was tritylated (+C(Ph3)) at the 5-position and a methanesulphonyl (+CH3SO2) group was introduced at the 3-OH group[5]. The methanesulphonyl group was substituted with iodine by refluxing the compound in 1,2-dimethoxythane in the presence of NaI. After chromatography on a silica column the 5-trityl-3-iodothymidine was hydrogenated in 80% acetic acid to remove the trityl group. The resultant 3-iodothymidine was hydrogenated to produce 23-dideoxythymidine which subsequently was phosphorylated. Once phosphorylated, ddTTP was then purified on a DEAE-sephadex column with triethylammonium-hydrogen carbonate gradient. Figure 3 is a schematic representation to produce ddT prior phosphorylation. When preparing ddGTP, the starting material was N-isobutyryl-5-O-monomethoxytrityldepxyguanosine [1]. After the tosylation of the 3-OH group the compound was then converted to the 23-didehydro derivative with sodium methoxide. Then the isobutyryl group was partly removed during this treatment of sodium methoxide and was removed completely by incubation in the presence of NH3 overnight at 45oC. During the overnight incubation period, the didehydro derivative was reduced to the dideoxy derivative and then converted to the triphosphate. The triphosphate was purified by the fractionation on a DEAE-Sephadex column using a triethylamine carbonate gradient. Figure 4 is a schematic representation to produce ddG prior phosphorylation. Preparing the ddCTP was similar to ddGTP, but was prepared from N-anisoyl-5-O-monomethoxytrityldeoxycytidine. However the purification process was omitted for ddCTP, as it produced a very low yield, therefore the solution was used directly in the experiment described in the paper [2]. Figure 5 is a schematic representation to produce ddC prior phosphorylation. With the four dideoxy samples now prepared, the sequencing procedure can now commence. The dideoxy samples are in separate tubes, along with restriction enzymes obtained from ?X174 replicative form and the four dNTPs [2]. The restriction enzymes and the dNTPs begin strand synthesis and the ddNTP is incorporated to the growing polynucleotide and terminates further strand synthesis. This is due to the lack of the hydroxyl group at the 3 position of ddNTP which prevents the next nucleotide to attach onto the strand. The four tubes are separate by gel-electrophoresis on acrylamide gels (see Gel-Electrophoresis). Figure 6 shows the sequencing procedure. Reading the sequence is straightforward [1]. The first band that moved the furthest is located, this represents the smallest piece of DNA and is the strand terminated by incorporation of the dideoxynucleotide at the first position in the template. The track in which this band occurs is noted. For example (shown in Figure 6), the band that moved the furthest is in track A, so the first nucleotide in the sequence is A. To find out what the next nucleotide, the next most mobile band corresponding to DNA molecule which is one nucleotide longer than the first, and in this example, the band is on track T. Therefore the second nucleotide is T, and the overall sequence so far is AT. The processed is carried on along the autoradiograph until the individual bands start to close in and become inseparable, therefore becoming hard to read. In general it is possible to read upto 400 nucleotides from one autoradiograph with this method. Figure 7 is a schematic representation of an autoradiograph. E ver since Sanger perfected the method of DNA sequencing, there have been advances methods of sequencing along with the achievements. Certain achievements such as the Human genome project and shall be discussed later on in this review. Gel-Electrophoresis Gel-Electrophoresis is defined as the movement of charged molecules in an electric field [1][8]. DNA molecules, like many other biological compounds carry an electric charge. With the case of DNA, this charge is negative. Therefore when DNA is placed in an electric field, they migrate towards the positive pole (as shown in figure 8). There are three factors which affect the rate of migration, which are shape, electrical charge and size. The polyacrylamide gel comprises a complex network of pores through which the molecules must travel to reach the anode. Maxam and Gilbert Method The Maxam and Gilbert method was proposed before Sanger Method in the same year. While the Sangers method involves enzymatic radiolabelled fragments from unlabelled DNA strands [2]. The Maxam-Gilbert method involves chemical cleavage of prelabelled DNA strands in four different ways to form the four different collections of labelled fragments [6][7]. Both methods use gel-electrophoresis to separate the DNA target molecules [8]. However Sangers Chain Termination method has been proven to be simpler and easier to use than the Maxam and Gilbert method [9]. As a matter of fact, looking through the literature text books, Sangers method of DNA sequencing have been explained rather than Maxam and Gilberts [1][3][9][10]. With Maxam and Gilberts method there are two chemical cleavage reactions that take place [6][7]. One of the chemical reaction take places with guanine and the adenine, which are the two purines and the other cleaves the DNA at the cytosine and thymin e, the pyrimidines. For the cleavage reaction, specific reagents are used for each of the reaction. The purine specific reagent is dimethyl sulphate and the pyrimidine specific reagent is hydrazine. Each of these reactions are done in a different way, as each of the four bases have different chemical properties. The cleavage reaction for the guanine/adenine involves using dimethyl sulphate to add a methyl group to the guanines at the N7 position and at the N3 position at the adenines [7]. The glycosidic bond of a methylated adenines is unstable and breaks easily on heating at neutral pH, leaving the sugar free. Treatment with 0.1M alkali at 90oC then will cleave the sugar from the neighbouring phosphate groups. When the resulting end-labelled fragments are resolved on a polyacrylamide gel, the autoradiograph contains a pattern a dark and light bands. The dark bands arise from the breakage at the guanines, which methylate at a rate which is 5-fold faster than adenines. From this reac tion the guanine appear stronger than the adenosine, this can lead to a misinterpretation. Therefore an Adenine-Enhanced cleavage reaction takes place. Figure 9 shows the structural changes of guanine when undergoing the structural modifications involved in Maxam-Gilbert sequencing. With an Adenine-Enhanced cleavage, the glycosidic bond of methylated adenosine is less stable than that of methylated guanosine, thus gentle treatment with dilute acid at the methylation step releases the adenine, allowing darker bands to appear on the autoradiograph [7]. The chemical cleavage for the cytosine and thymine residues involves hydrazine instead of dimethyl sulphate. The hydrazine cleaves the base and leaving ribosylurea [7]. After partial hydrazinolysis in 15-18M aqueous hydrazine at 20oC, the DNA is cleaved with 0.5M piperidine. The piperidine (a cyclic secondary amine), as the free base, displaces all the products of the hydrazine reaction from the sugars and catalyzses the b-elimination of the phosphates. The final pattern contains bands of the similar intensity from the cleavages at the cytosines and thymines. As for cleavage for the cytosine, the presence of 2M NaCl preferentially suppresses the reaction of thymine with hydrazine. Once the cleavage reaction has taken place each original strand is broken into a labelled fragment and an unlabelled fragment [7]. All the labelled fragments start at the 5 end of the strand and terminate at the base that precedes the site of a nucleotide along the original strand. Only the labelled fragmen ts are recorded on the gel electrophoresis. Dye-labelled terminators For many years DNA sequencing has been done by hand, which is both laborious and expensive[3]. Before automated sequencing, about 4 x 106 bases of DNA had been sequenced after the introduction of the Sangers method and Maxam Gilbert methods [11]. In both methods, four sets of reactions and a subsequent electrophoresis step in adjacent lanes of a high-resolution polyacrylamide gel. With the new automated sequencing procedures, four different fluorophores are used, one in each of the base-specific reactions. The reaction products are combined and co-electrophoresed, and the DNA fragments generated in each reaction are detected near the bottom of the gel and identified by their colour. As for choosing which DNA sequencing method to be used, Sangers Method was chosen. This is because Sangers method has been proven to be the most durable and efficient method of DNA sequencing and was the choice of most investigators in large scale sequencing [12]. Figure 10 shows a typical sequence is ge nerated using an automated sequencer. The selection of the dyes was the central development of automated DNA sequencing [11]. The fluorophores that were selected, had to meet several criteria. For instance the absorption and emission maxima had to be in the visible region of the spectrum [11] which is between 380 nm and 780 nm [10], each dye had to be easily distinguishable from one another [11]. Also the dyes should not impair the hybridisation of the oligonucleotide primer, as this would decrease the reliability of synthesis in the sequencing reactions. Figure 11 shows the structures of the dyes which are used in a typical automated sequencing procedure, where X is the moiety where the dye will be bound to. Table 1 shows which dye is covalently attached to which nucleotide in a typical automated DNA sequencing procedure Dye Nucleotide Attached Flourescein Adenosine NBD Thymine Tetramethylrhodamine Guanine Texas Red Cytosine In designing the instrumentation of the florescence detection apparatus, the primary consideration was sensitivity. As the concentration of each band on the co-electrophoresis gel is around 10 M, the instrument needs to be capable of detecting dye concentration of that order. This level of detection can readily be achieved by commercial spectrofluorimeter systems. Unfortunately detection from a gel leads to a much higher background scatter which in turn leads to a decrease in sensitivity. This is solved by using a laser excitation source in order to obtain maximum sensitivity [11]. Figure 12 is schematic diagram of the instrument with the explanation of the instrumentation employed. When analyzing data, Hood had found some complications [11]. Firstly the emission spectra of the different dyes overlapped, in order to overcome this, multicomponent analysis was employed to determine the different amounts of the four dyes present in the gel at any given time. Secondly, the different dye molecules impart non-identical electrophoretic mobilities to the DNA fragments. This meant that the oligonucleotides were not equal base lengths. The third major complication was in analyzing the data comes from the imperfections of the enzymatic methods, for instance there are often regions of the autoradiograph that are difficult to sequence. These complications were overcome in five steps [11] High frequency noise is removed by using a low-pass Fourier filter. A time delay (1.5-4.5 s) between measurements at different wavelength is partially corrected for by linear interpolation between successive measurements. A multicomponent analysis is performed on each set of four data points; this computation yields the amount of each of the four dyes present in the detector as a function of time. The peaks present in the data are located The mobility shift introduced by the dyes is corrected for using empirical determined correction factors. Since the publication of Hoods proposal of the fluorescence detection in automated DNA sequence analysis. Research has been made on focussed on developing which are better in terms of sensitivity [12]. Bacterial and Viral Genome Sequencing (Shotgun Sequencing) Prior to 1995, many viral genomes have been sequenced using Sangers chain termination technique [13], but no bacterial genome has been sequenced. The viral genomes that been sequenced are the 229 kb genome of cytomegalovirus [14], and the 192 kb genome of vaccinia [15], the 187 kb mitochondrial and 121 kb cholorophast genomes of Marchantia polymorpha have been sequenced [16]. Viral genome sequencing has been based upon the sequencing of clones usually derived from extensively mapped restriction fragments, or ? or cosmid clones [17]. Despite advances in DNA sequencing technology, the sequencing of genomes has not progressed beyond clones on the order of the size of the ~ 250kb, which is due to the lack of computational approaches that would enable the efficient assembly of a large number of fragments into an ordered single assembly [13][17]. Upon this, Venter and Smith in 1995 proposed Shotgun Sequencing and enabled Haemophilus influenzae (H. influenzae) to become the first bacterial genome to be sequenced [13][17]. H. influenzae was chosen as it has a similar base composition as a human does with 38 % of sequence made of G + C. Table 2 shows the procedure of the Shotgun Sequencing [17]. When constructing the library ultrasonic waves were used to randomly fragment the genomic DNA into fairly small pieces of about the size of a gene [13]. The fragments were purified and then attached to plasmid vectors[13][17]. The plasmid vectors were then inserted into an E. coli host cell to produce a library of plasmid clones. The E. coli host cell strains had no restriction enzymes which prevented any deletions, rearrangements and loss of the clones [17]. The fragments are randomly sequenced using automated sequencers (Dye-Labelled terminators), with the use of T7 and SP6 primers to sequence the ends of the inserts to enable the coverage of fragments by a factor of 6 [17]. Table 2 (Reference 17) Stage Description Random small insert and large insert library construction Shear genomic DNA randomly to ~2 kb and 15 to 20 kb respectively Library plating Verify random nature of library and maximize random selection of small insert and large insert clones for template production High-throughput DNA sequencing Sequence sufficient number of sequences fragments from both ends for 6x coverage Assembly Assemble random sequence fragments and identity repeat regions Gap Closure Physical gaps Order all contigs (fingerprints, peptide links, ÃŽ », clones, PCR) and provide templates for closure Sequence gaps Complete the genome sequence by primer walking Editing Inspect the sequence visually and resolve sequence ambiguities, including frameshifts Annotation Identify and describe all predicted coding regions (putative identifications, starts and stops, role assignments, operons, regulatory regions) Once the sequencing reaction has been completed, the fragments need to be assembled, and this process is done by using the software TIGR Assembler (The Institute of Genomic Research) [17]. The TIGR Assembler simultaneously clusters and assembles fragments of the genome. In order to obtain the speed necessary to assemble more than 104 fragments [17], an algorithm is used to build up the table of all 10-bp oligonucleotide subsequences to generate a list of potential sequence fragment overlaps. The algorithm begins with the initial contig (single fragment); to extend the contig, a candidate fragment is based on the overlap oligonucleotide content. The initial contig and candidate fragment are aligned by a modified version of the Smith-Waterman [18] algorithm, which allows optional gapped alignments. The contig is extended by the fragment only if strict criteria of overlap content match. The algorithm automatically lowers these criteria in regions of minimal coverage and raises them in r egions with a possible repetitive element [17]. TIGR assembler is designed to take advantage of huge clone sizes [17]. It also enforces a constraint that sequence from two ends of the same template point toward one another in the contig and are located within a certain range of the base pair [17]. Therefore the TIGR assembler provides the computational power to assemble the fragments. Once the fragments have been aligned, the TIGR Editor is used to proofread the sequence and check for any ambiguities in the data [17]. With this technique it does required precautionary care, for instance the small insert in the library should be constructed and end-sequenced concurrently [17]. It is essential that the sequence fragments are of the highest quality and should be rigorously check for any contamination [17]. Pyrosequencing Most of the DNA sequencing required gel-electrophoresis, however in 1996 at the Royal Institute of Technology, Stockholm, Ronaghi proposed Pyrosequencing [19][20]. This is an example of sequencing-by-synthesis, where DNA molecules are clonally amplified on a template, and this template then goes under sequencing [25]. This approach relies on the detection of DNA polymerase activity by enzymatic luminometric inorganic pyrophosphate (PPi) that is released during DNA synthesis and goes under detection assay and offers the advantage of real-time detection [19]. Ronaghi used Nyren [21] description of an enzymatic system consisting of DNA polymerase, ATP sulphurylase and lucifinerase to couple the release of PPi obtained when a nucleotide is incorporated by the polymerase with light emission that can be easily detected by a luminometer or photodiode [20]. When PPi is released, it is immediately converted to adenosine triphosphate (ATP) by ATP sulphurylase, and the level of generated ATP is sensed by luciferase-producing photons [19][20][21]. The unused ATP and deoxynucleotide are degraded by the enzyme apyrase. The presence or absence of PPi, and therefore the incorporation or nonincorporation of each nucleotide added, is ultimately assessed on the basis of whether or not the photons are detected. There is minimal time lapse between these events, and the conditions of the reaction are such that iterative addition of the nucleotides and PPi detection are possible. The release of PPi via the nucleotide incorporation, it is detected by ELIDA (Enzymatic Luminometric Inorganic pyrophosphate Detection Assay) [19][21]. It is within the ELIDA, the PPi is converted to ATP, with the help of ATP sulfurylase and the ATP reacts with the luciferin to generate the light at more than 6 x 109 photons at a wavelength of 560 nm which can be detected by a photodiode, photomultiplier tube, or charge-coupled device (CCD) camera [19][20]. As mentioned before, the DNA molecules need to be amplified by polymerase chain reaction (PCR which is discussed later Ronaghi observed that dATP interfered with the detection system [19]. This interference is a major problem when the method is used to detect a single-base incorporation event. This problem was rectified by replacing the dATP with dATPaS (deoxyadenosine a–thiotrisulphate). It is noticed that adding a small amount of the dATP (0.1 nmol) induces an instantaneous increase in the light emission followed by a slow decrease until it reached a steady-state level (as Figure 11 shows). This makes it impossible to start a sequencing reaction by adding dATP; the reaction must instead be started by addition of DNA polymerase. The signal-to-noise ratio also became higher for dATP compared to the other nucleotides. On the other hand, addition of 8 nmol dATPaS (80-fold higher than the amount of dATP) had only a minor effect on luciferase (as Figure 14 shows). However dATPaS is less than 0.05% as effective as dATP as a substrate for luciferase [19]. Pyrosequencing is adapted by 454 Life Sciences for sequencing by synthesis [22] and is known as the Genome Sequencer (GS) FLX [23][24]. The 454 system consist of random ssDNA (single-stranded) fragments, and each random fragment is bound to the bead under conditions that allow only one fragment to a bead [22]. Once the fragment is attached to the bead, clonal amplification occurs via emulsion. The emulsified beads are purified and placed in microfabricated picolitre wells and then goes under pyrosequencing. A lens array in the detection of the instrument focuses luminescene from each well onto the chip of a CCD camera. The CCD camera images the plate every second in order to detect progression of the pyrosequencing [20][22]. The pyrosequencing machine generates raw data in real time in form of bioluminescence generated from the reactions, and data is presented on a pyrogram [20] Sequencing by Hybridisation As discussed earlier with chain-termination, Maxamm and Gilbert and pyrosequencing, these are all direct methods of sequencing DNA, where each base position is determined individually [26]. There are also indirect methods of sequencing DNA in which the DNA sequence is assembled based on experimental determination of oligonucleotide content of the chain. One promising method of indirect DNA sequencing is called Sequencing by Hybridisation in which sets of oligonucleotide probes are hybridised under conditions that allow the detection of complementary sequences in the target nucleic acid [26]. Sequencing by Hybridisation (SBH) was proposed by Drmanac et al in 1987 [27] and is based on Dotys observation that when DNA is heated in solution, the double-strand melts to form single stranded chains, which then re-nature spontaneously when the solution is cooled [28]. This results the possibility of one piece of DNA recognize another. And hence lead to Drmanac proposal of oligonucleotides pro bes being hybridised under these conditions allowing the complementary sequence in the DNA target to be detected [26][27]. In SBH, an oligonucleotide probe (n-mer probe where n is the length of the probe) is a substring of a DNA sample. This process is similar to doing a keyword search in a page full of text [29]. The set of positively expressed probes is known as the spectrum of DNA sample. For example, the single strand DNA 5GGTCTCG 3 will be sequenced using 4-mer probes and 5 probes will hybridise onto the sequence successfully. The remaining probes will form hybrids with a mismatch at the end base and will be denatured during selective washing. The five probes that are of good match at the end base will result in fully matched hybrids, which will be retained and detected. Each positively expressed serves as a platform to decipher the next base as is seen in Figure 16. For the probes that have successfully hybridised onto the sequence need to be detected. This is achieved by labelling the probes with dyes such as Cyanine3 (Cy3) and Cyanine5 (Cy5) so that the degree of hybridisation can be detected by imaging devices [29]. SBH methods are ideally suited to microarray technology due to their inherent potential for parallel sample processing [29]. An important advantage of using of using a DNA array rather than a multiple probe array is that all the resulting probe-DNA hybrids in any single probe hybridisation are of identical sequence [29]. One of main type of DNA hybridisation array formats is oligonucleotide array which is currently patented by Affymetrix [30]. The commercial uses of this shall be discussed under application of the DNA Array (Affymetrix). Due to the small size of the hybridisation array and the small amount of the target present, it is a challenge to acquire the signals from a DNA Array [29]. These signals must first be amplified b efore they can be detected by the imaging devices. Signals can be boosted by the two means; namely target amplification and signal amplification. In target amplification such as PCR, the amount of target is increased to enhance signal strength while in signal amplification; the amount of signal per unit is increased. Nanopore Sequencing Nanopore sequencing was proposed in 1996 by Branton et al, and shows that individual polynucleotide molecules can be characterised using a membrane channel [31]. Nanopore sequencing is an example of single-molecule sequencing, in which the concept of sequencing-by-synthesis is followed, but without the prior amplification step [24]. This is achieved by the measurement of ionic conductance of a nucleotide passing through a single ion channels in biological membranes or planar lipid bilayer. The measurement of ionic conductance is routine neurobiology and biophysics [31], as well as pharmacology (Ca+ and K+ channel)[32] and biochemistry[9]. Most channels undergo voltage-dependant or ligand dependant gating, there are several large ion channels (i.e. Staphylococcus aureus a-hemolysin) which can remain open extended periods, thereby allowing continuous ionic current to flow across a lipid bilayer [31]. If a transmembrane voltage applied across an open channel of appropriate size should d raw DNA molecules through the channel as extended linear chains whose presence would detect reduce ionic flow. It was assumed, that the reduction in the ionic flow would lead to single channel recordings to characterise the length and hence lead to other characteristics of the polynucleotide. In the proposal by Branton, a-hemolysin was used to form a single channel across a lipid bilayer separating two buffer-filled compartment [31]. a-Hemolysin is a monomeric, 33kD, 293 residue protein that is secreted by the human pathogen Staphylococcus aureus [33]. The nanopore are produced when a-hemolysin subsunits are introduced into a buffered solution that separates lipid bilayer into two compartments (known as cis and trans): the head of t

Autonomous Vehicle Parking Using Finite State Automata Information Technology Essay

Autonomous Vehicle Parking Using Finite State Automata Information Technology Essay Our project is based on the autonomous parking using finite state automata. The invention of the autonomous parking system in which the elevator, lifter and computer are connected in group such that each unit swap information as it require and then the system calculating processes of loading and unloading a car, issuing the parking ticket and then identifying the parking ticket. The parking system include a elevator , lifter, computer, database for storing vehicle number data and information data which only display that how many car are parking in different floors and then elevator load car , park in vacant floor and unloaded the car one by one and. The process of elevator is to pick and lift the car from ground to parking area and then back to it owns position. The present invention narrates to a system of controlling a autonomous parking system and calculating a parking fee and more principally to a parking control system to which a computer, a control unit, a parking ticket issuer, and a parking ticket recognizer are connected through a network for allowing any required information to be used among them. Description of the Prior Art Generally in a conventional autonomous parking system, a car is moved to a respective floor along hoist way with use of a lift, and then parked in respective parking space with use of pallets. In that system, a fee calculator and a parking ticket issuer are separately operated such that each operation of a parking machine, a parking ticket issuer, and the fee calculator is performed separately. History The parking of vehicles in the employment center of larger cities of the world has increasingly become a major problem. Not only is there insufficient available land for surface parking but the high cost of the land makes such a use economically infeasible. In addition, the aggregation of a large amount of vehicles causes traffic, environmental, aesthetic, and pollution problems. The solution to locate parking facilities in more remote areas where land cost are lower is also not feasible because of inconvenient distances to areas of employment and potential safety and security problem To construct large underground parking facility in these congested center city areas also create major problem because of the high cost of the instruction the multitude of underground utility encountered and the inevitable distribution to existing services during the construction period This complex situation indicates that the use of small low volume underground parking facility with the capability of the fitting within the existing infra structure would be highly desirable. Not only would be they be able to be located conveniently to the drivers destination but they also have potential to maintain the surface above the parking facility in a park Other generators that demand more convenient, close or adjacent parking solution include office buildings, apartments, hotels and institutions where surface parking would not meet the requirements by reason of restricted land area, high land costs, inconvenient access, security, and environmental factors. In response to these needs for alternative and convenient parking, the engineering group endeavored to engineer a solution. They had previously solved couples design challenges in the construction industry by utilizing an approach that included standardization and variety reduction in order to reduce construction costs and time and to provide greater consumer benefit. Their solution was autonomous parking system, a modular automated parking system that could be installed below or above ground, alone or in repeat modules. This approach provided the advantages of minimizing inconvenience, expediting construction time, and lowering construction cost. Its compact area permitted it to be built in center city areas, while avoiding the problems of interference with dense underground utilities and major disruptions to these services. INTRODUCTION Autonomous parking is an autonomous car planning from a line of traffic into a parking place to perform parallel parking. The autonomous parking intends to develop the ease and safety of driving in controlled situations where much attention and knowledge is required to steer the vehicle. The parking scheme is achieved by means of synchronized control of the steering angle and speed which takes into account the actual circumstance in the environment to make sure collision-free motion within the available space. OR The automated vehicle parking system for a parking facility that be in handle with a vehicle approaching or leaving the facility with RF signals, or the like, that identify the vehicle and sends the vehicle identification number, time of day, and lane number to a central computer for calculating the parking cost based on rates for each individual vehicle stored in the computer. [7] EVOLUTIONARY FUNCTIONAL TEST OF THE AUTONOMOUS PARKING SYSTEM THE AUTONOMOUS PARKING SYSTEM As an automobile manufacturer, DaimlerChrysler is continuously developing new systems in order to improve vehicle safety, quality, and comfort. Within this context, prototypical vehicle systems are developed, which support autonomous vehicle parking a function that might be introduced to the market in some years time. The autonomous parking systems regarded in this paper are intended to automate parking lengthways into a parking space, like shown in Fig.1. For this purpose, the vehicle is equipped with environmental sensors, which register objects surrounding the vehicle. On passing along, the system can recognize sufficiently large parking spaces and can signal to the driver that a parking space has been found. If the driver decides to park in the vehicle can do this automatically. Fig 1: Functionality of Autonomous Parking System [1] In Fig.2 the system environment for the autonomous parking system is shown. The inputs are sensor data, which contain information on the state of the vehicle, e.g. vehicle speed or steering position, and information from the environmental sensors, which register objects on the left and right hand side of the vehicle. For output the system possesses an interface to the vehicle actors, where the vehicles velocity and steering angle will be set. The internal structure of the autonomous parking Fig 2: System Environment- [2] The parking space detection processes the data from the environmental sensor systems and delivers the recognized geometry of a parking space if it has been detected to be sufficiently large. The parking controller component uses the geometry data of the parking space together with the data from the vehicle sensors to steer the vehicle through the parking procedure. For this purpose, velocity and steering angle are set for the vehicle actors. Fig 3: Subcomponents of Autonomous Parking System [3] Some proposals for solving of parking problems A good decision is to be built automated parking systems for cars preferably served by stacker cranes (see fig 4), that are the basic element of the automated warehouse structures. Fig 4: [4] Brief Description about FINITE STATE AUTOMATA Finite-State Automata A finite-state transducer whose output components are ignored is called a finite-state automaton. Formally, a  finite-state  automaton  M is a tuple , where Q,  , q0, and F are defined as for finite-state transducers, and the transition table  Ã‚  is a relation from Q ÃÆ'- (  Ã‚  {}) to Q. Kinds of Finite State Automata 1. DFA 2. NFA What is NFA? In the theory of computation, nondeterministic finite automaton (NFA)  is a  finite state machine  where for each pair of state and input symbol there may be several possible next states. This distinguishes it from the  deterministic finite automaton  (DFA), where the next possible state is uniquely determined. Although the DFA and NFA have distinct definitions, it may be shown in the formal theory that they are equivalent, in that, for any given NFA, one may construct an equivalent DFA, and vice-versa: this is the  power set construction. Both types of automata recognize only  regular languages. Non-deterministic finite state machines are sometimes studied by the name  sub shifts of finite type. Non-deterministic finite state machines are generalized by  probabilistic automata, which assign a probability to each state transition. Formal Definition Two similar types of NFAs are commonly defined: the NFA and the  NFA with ÃŽÂ µ-moves. The ordinary NFA is defined as a  5-tuple, (Q, ÃŽÂ £,  T,  q0, F), consisting of a finite  set  of states  Q a finite set of  input symbols  ÃƒÅ½Ã‚ £ a transition  function  T  :  Q  ÃƒÆ'- ÃŽÂ £ à ¢Ã¢â‚¬  Ã¢â‚¬â„¢Ã‚  P(Q). an  initial  (or  start) state  q0  Ãƒ ¢Ã‹â€ Ã‹â€ Ã‚  Q a set of states  F  distinguished as  accepting  (or  final)  states  F  Ãƒ ¢Ã…  Ã¢â‚¬  Ã‚  Q. What is DFA? In the  theory of computation, a  deterministic finite state machine-also known as  deterministic finite state automaton  (DFSA) is a  finite state machine  where for each pair of state and input symbol there is one and only one transition to a next state, as opposed to a  nondeterministic finite-state machine, which has the possibility of multiple transitions . DFAs recognize the set of  regular languages  and no other languages. A DFA will take in a string of input symbols. For each input symbol it will then transition to a state given by following a transition function. When the last input symbol has been received it will either accept or reject the string depending on whether the DFA is in an accepting state or a non-accepting state. Formal Definition A DFA is a 5-tuple, (Q, ÃŽÂ £, ÃŽÂ ´,  q0,  F), consisting of a finite set of  states  (Q) a finite set of input symbols called the  alphabet  (ÃŽÂ £) a transition  function  (ÃŽÂ ´Ã‚  :  Q  ÃƒÆ'- ÃŽÂ £ à ¢Ã¢â‚¬  Ã¢â‚¬â„¢Ã‚  Q) a  start state  (q0  Ãƒ ¢Ã‹â€ Ã‹â€ Ã‚  Q) a set of  accept states  (F  Ãƒ ¢Ã…  Ã¢â‚¬  Ã‚  Q) Finite State Machine of Autonomous Vehicle parking Language (L) = {pick car, first floor, second floor, third floor, go to, back, ground, elevator} Third Floor Second Floor First Floor Elevator Ground stand Back into position goto Pick Car back goto goto goto Back back goto goto Fig 5 Finite State Machine of Autonomous Vehicle parking NFA to DFA Finite State Machine of Autonomous Vehicle parking Let assigned digit code to each statement in above machine. e d c b a 0 0 1 0 1 1 1 0 0 1 1 Tabular Form S x I 0 1 a A B b A cde c A D d C E e D error cde Acd de acd Ac bde de Cd E ac A bd bde Acd cde cd Ac de bd Ac cde b a cde cd de ac bde DFA Machine 0 0 1 0 1 0 acd 1 1 1 0 0 0 bd 1 0 1 1 0 0 Q={a,b,c,d,e} q0={a} qf={a,c,d,e} Language= {0,1} S=Q X I S= (a, 0) = a S= (a, 1) = b S= (b, 0) = a S= (b, 1) = cde S=(c, 0) = a S=(c, 1) = d S= (d, 0) = c S= (d, 1) = e S= (e, 0) = d S= (e, 1) =error Autonomous Parking Solutions Autonomous parking solutions are capable to way store huge number of vehicles within sufficient space. How Autonomous Parking System works The procedure of autonomous parking solution begins as soon as elevator come to pick the car, the door I s then closed and after that stored into vacant parking space and automatically return to entry box when we press the button. Maximizing the use of space for parking Besides the ease of parking for the user, maximum parking capacity is guaranteed because there are no slopes of carriageways with the autonomous parker. Reasons why Autonomous Parking System is the ideal solution There are several reason why autonomous park is the ideal solution because whenever and wherever the huge number of vehicle park need to be park over the available space, e.g. optimum access times, comfortable operation, protection against theft, robbery, burglary and harm, low maintenance requirements, and last but not least, high adaptability to the individual garage planning project with respect to the options regarding maximum possible car heights. [6] Automated Parking General Descriptions Below is a list of terms and definitions to better assist you in understanding the nomenclature in the FATA Sky parks automated parking systems. Entry section The point where the driver parks their vehicle. Exit section The point where the driver retrieves their vehicle and where the APS will transport the vehicle when the request is made. Joint Entry and Exit The vehicle is dropped off and picked up at the same section. This option requires extra space allow for a turntable to turn the vehicle around. Turntable Speeds up the retrieval time by simplifying the exit system in combination sections. The vehicle is turned 180 degrees and is ready to be driven forward out of the Combined Entry/Exit module, rather than being backed out. Robot Shuttle The mechanical part of the system that picks up the parked vehicle in the entry/exit or combo section and moves the vehicles horizontally along the primary walkway to a vertical lift or available parking space. Multiple robots can be used. Dedicated Robots Robot shuttles that are dedicated to each parking floor. benefit once the vehicle is put down on a vertical lift the robot can retrieve another call on that level. The dedicated choice is generally a faster system. Roaming Robots Robot shuttles that travel through the system with the vehicles on them by riding up on a end-of-aisle lift. Benefit less robots are used, but release times are compact. Autonomous The robots move separately from each other. Advantage if a robot requires service the efficiency of the system is only modestly affected. Off-Corridor Vertical Lift The portion of the system that moves the vehicles vertically from the entry level to an above/below ground-parking floor. Used in conjunction with dedicated robot shuttles and only transfers the vehicle to alternate floors. Typically used on systems that have a robot aisle greater than 75-100 in length to increase delivery speeds. End-of-aisle Vertical Lift The section of the system that moves the vehicles vertically from the entry level to an above/below ground-parking floor. Used in combination with Roaming robot transports and lifts the robot carry and vehicle to alternate floors. Typically used for systems with a robot aisle less than 100 in length. Layout The organization of the parking structure including the demonstration of parked vehicles off of the robot aisle. Conventional parking pass on to the typical concrete parking structures with vehicle slope access to multiple floors. Non-Automated Queuing Time Queuing time refers to the time necessary the system is busy before another vehicle is allowed to enter the system. Queuing time is dependent relative on the entry and exit time of the driver (length of time to depart or enter the parked car) and the amount of automated lifts, robots, and entry / exit modules utilized. Attendant Requirements No attendant is required, however, an attendant is useful in assisting drivers to negotiate the system. If the parking lot is open to the public and not strictly the building tenants, an attendant is recommended. Benefit of parking Guidance System/Autonomous Parking System The obvious benefit of automated parking systems  is the ability to fit more cars in less space which can solve many parking problems, but there are many other benefits to the developer, operator,  consumer and society in general.   For example, consider the countless acres  of open space consumed by  parking lots  and all the storm water runoff generated by that entire impervious surface.   Self park ramp garages  are more efficient, but still take twice the space as an automated parking facility.     By contrast, automated parking garages are a green solution since they  preserve open space,  have low energy consumption and have no carbon emissions, because vehicles are shut off before being parked in the system. General benefits Decrease in time spent for searching parking. The efficiency and accessibility benefits from reduced searching can also cause good result in some lessening in accidents due to reduced driver frustration Reduced pollution. Changes in pollutant emissions due to Parking Guidance information are most closely related to changes in overall travel time, for example, yearly pollutant release are reported to have been reduced due to a PGI system in Munich, Germany. Reduction in traffic jams due to fewer cars driving around for spaces searching.   Elimination of stand in line entering parking facilities because drivers will not go to a facility where there is no available space.   Reduction in unlawfully parked vehicles.   Better distribution of flow and parking demand through the area. Autonomous parking systems result in higher revenues and profitability for the parking facilities.   Operator Benefits    Reduced labor    Reduced liability    Reduced lighting HVAC      Total control over access enforcement Consumer Social Benefits    Eliminate fender benders    Eliminate theft and vandalism    Reduce carbon emissions    Preserve open space Highlights of autonomous parking The main highlights of the automated parking systems served by stacker crane in comparison with other systems are: optimum use of the available space, minimum room of a parking automobile, no need of platforms and staircases, module principle of building, that meets the requirements of the clients, shortening the time needed for building a parking system, Less time for parking and forwarding because of the high traveling speed of the stacker crane and simultaneously vertical and horizontal movement and so on. Examples of Autonomous Parking System Features of Hoboken, New Jersey Benefits of the autonomous parking system include: optimization of space utilization, security, convenience, lower garage owners liability insurance, greater reduction schedule, lower lighting and ventilation requirements (no cars driving around inside; no people go inside), and lower emissions and less pollution (clean parking system). Car Towers at the Autostadt: A Hive for Beetles Fig [5] Features The Car Towers is a 20-story tall car storage space tower in Wolfsburg, Germany. Its owned operated by Volkswagen, which enlighten why all the cars around 800 at full capacity are VWs. The Car Towers has often been used to demonstrate public parking garages of the future even though its a private endeavor that merely allows VW to save space. Dubai Robotic Car park Fig - [8] Features The robotic car park in Deira, Dubai (above) doesnt have the enough storage space of the Car Towers (14 cars instead of 800) but it is practical, workable and open for business. A 67-car capacity robot car park in New Yorks Chinatown works on the same principal. CONCLUSIONS The future belongs to the automated parking garages and the efforts made in this direction account for the investments. Automated parking garages are better decision from the social point of view. For example the owners of cars who park their cars in parking automat are forced to pay higher parking charges. This is extremely important in order to shorten the time for servicing a single cell for parking, which is a basic criteria in building automated parking systems for cars. Summary The primary purpose of this autonomous parking study was to determine short-term and long-term recommendations to improve parking in cities. The parking study initially evaluated existing conditions, determined primarily through reviews of background materials (including previous parking studies), . The examination of existing conditions provided the baseline data from which future development, with its impact on parking supply and demand, could be evaluated. Finally, parking alternatives were considered to address future needs, as well as improve the utilization and efficiency of existing parking resources. Future parking alternatives included potential parking supply changes, as well as general parking management strategies.

The Importance of Education in Preventing Prejudice Essays -- Importan

Ignorance is a huge problem, it is one of the biggest factors responsible for issues such as racism and sexism. Luckily, ignorance, generally speaking, is a relatively easy issue to fix. The obvious answer here would be more education, but this is not necessarily the case. In order to eliminate much of the racism, sexism, and other forms of prejudice that arise due to ignorance, it is necessary to look at education from another perspective: one that encourages togetherness and development alongside people of all races and genders. One quote by Grace Boggs book The Next American Revolution summarizes the issue perfectly. â€Å"Just imagine what our neighborhoods would be like if, instead of keeping our children isolated in classrooms for twelve years and more, we engaged them in community-building activities with the same audacity with which the civil rights movement engaged them in desegregation activities fifty years ago! ...Our children will be absorbing naturally and norm ally the values of social responsibility and cooperation at the same time that they are being inspired to learn the skills and acquire the information necessary to solve real problems† (Boggs 158). So, the main point here is that prejudice, against all sexes, genders, and creeds, can be eliminated via education that encourages cooperation with the largest variety of people. In order to understand this concept, it is necessary to look at it from a few different perspectives to analyze its viability in modern society. First, it is necessary to examine the current paradigm within education in order to determine exactly what it is about the modern system that requires changing. One of the most immediate concerns comes as a result of the school facilities the... ...r through the power of music, will not be forgotten. Racism, sexism, and other forms of prejudice are simply not acceptable in modern society, and the sooner they can be eliminated altogether, the sooner the world can truly advance. Works Cited Boggs, Grace Lee, and Scott Kurashige. The next American revolution: Sustainable activism for the twenty-first century. University of California Pr, 2012. Print. Dyson, Michael Eric. Holler If You Hear Me 2006: Searching for Tupac Shakur. Basic Civitas Books, 2006. Print. Godin, Seth. Stop stealing dreams. 2012. Print. Hooks, Bell. Teaching to transgress: Education as the practice of freedom. Vol. 4. New York: Routledge, 1994. Print. Marx, Karl, and Friedrich Engels. Manifesto of the communist party. CH Kerr & Company, 1906. Print. Tupac: Resurrection Dir. Lauren Lazin. Perf. Tupac Shakur. 2003. Film.

Merchant of Venice Essay: Refuting the Critics -- Merchant Venice Ess

In The Jew of Venice, Granville takes up and refutes the principal "subversions," in The Merchant of Venice that modern and postmodern critics have imposed upon on the play. Without its’ alleged contradictions, the play has a tight formalist structural unity, it focuses on an essentialist Platonic idea, and, resolving all conflicts, it ends in closure. On the topic of Antonio's sadness, Granville picks up a clue that to my knowledge no modern critic has noticed. In his "methodizing" process, he moved Antonio's play-opening line--"I know not why I am so sad"--to Bassan- io's feast, between the toasts and the masque, and merged it with Jessica's fifth act misgiving--"I am never merry when I hear sweet music" (5.1.69). Listening to the music at his friend's feast, Granville's Antonio laments, O Bassanio! There sits a heaviness upon my heart Which wine cannot remove: I know not But music ever makes me thus. (2.2.35-38) Lorenzo's comforting answer to Jessica in act 5 of Shakespeare's play then becomes Bassanio's comforting answer to Antonio act 2 of Granville's: The reason is, your spirits are attentive: &nb... ... spoils." In The Jew of Venice, Granville, who resides in Shakespeare's own moral community, takes up and refutes the principal "subversions," "leaks," "interrogations," and "dark shadows" in The Merchant of Venice that modern and postmodern critics, working from what I argue are irrelevant post- capitalist prejudices, have imposed upon on the play. Without its’ alleged contradictions, the play has a tight formalist structural unity, it focuses on an essentialist Platonic idea, and, resolving all conflicts, it ends in closure. Unless there are other reasons than those commonly given for alleging that The Merchant of Venice is "multivalent and "plural" in meaning, we will have to assume, for the time being at least, that it isn't.

A Day In The Life Of A Gnome :: essays research papers

A Day in the Life of a Gnome Once upon a time there was a gnome named Knob, who lived the far off land of Gnomania. Gnomania is a huge underground lair with only one entrance. The only entrance to this lair is by swimming down to the bottom of the ocean floor and knocking on a large clam. On the other side of this clam is an old grouchy gnome named Stubby whose only job was to keep the clam clean, and listen for the knock of incoming visitors. To assist Stubby in guarding the clam, he had a pet snark. A snark is an animal or pet that can be used as a watchdog. One day Knob went to visit his grandmother in the land of Gnollie which is about a guzillion centimeters away from Gnomania. On his way to his grandmother's house he ran across a garden. Upon stumbling on the garden, he decided to be thoughtful and pick some tulips for his grandmother. Tulips were her favorite snack. While approaching his grandmother's house, he found something to be very strange. His grandmother was hovering on an hummingbird, while picking apples from her tall twenty-foot appletree. After contemplating on this strange doing, he decided not to let it bother him. After seeing Knob approach her house, Knob's grandmother whistled for the hummingbird to bring her down so she could greet him. She greeted him with a loud burp which was a common courtesy among Gnomanians. After greeting him she also sprinkled him with some of her fairy dust. She handily carried it around in her turban. She naturally invited him in for dinner where they ate lots of mosquitoes. After dinner, they watched a cheap imitation of television. They used a big box to put different animals in and called it the Discovery channel. By the end of the day Knob was getting a little weary, so he decided to return home. When he returned form Gnollie his pet snark was there to welocme him home. He welcomed his snark back by feeding it fisheyes or to us humans dog biscuits. After the short snack, Knob and his snark went back to his teepee where they hibernated until the next morning. The next day Knob met up with his friend Door. Door was a runaway gnome who lived on the streets of Gnomania. They met one day by accident. Since Door lived on the streets, he had to steal food to survive. One day when Door was running away from a gardener he ran full speed into Knob.

How Lennie Is Portrayed in the First Chapter Essay

To begin with, Steinbeck describes Lennie’s physical features to be very animal-like. He states that he walked â€Å"the way a bear drags his paws†. The use of animal imagery in this quote illustrates an image of a huge man heavily dragging himself, not only suggesting his size but also suggesting his immense strength. Due to his physicality, he is unable to carry out tasks normal people would be able to do. He is unable to control himself, specifically his strength—thus easily breaking things. Steinbeck emphasizes this as he brings up incidents of Lennie having killed mice and other small, fragile animals, while petting them. Lennie is also described to have an animal’s mental state, leaning towards animal instincts rather than human instincts. Steinbeck states he’d â€Å"drink out of a gutter if [he] was thirsty†, displaying Lennie’s rash behaviour. His urge to satisfy his immediate desire would overpower the need to be cautious and aware of his surroundings. Many people think before they act. In Lennie’s case however, he doesn’t think at all as George does it for him. Lennie was â€Å"snorting into the water like a horse† which also proves his uncivilized manner. Furthermore, Lennie’s animal instincts prevent him from being able to learn. Like wild animals, he is uncontrollable and unpredictable; though he may be trained and taught not to do some things, they are bound to commit the same mistakes over and over again. Steinbeck states that George had â€Å"hopelessly† warned Lennie about the water he was drinking, proving that George’s warnings will not affect Lennie in any way as he is bound to forget—he will never learn. Not only is Lennie’s mental behaviour similar to an animal, but it is also similar to a child. Lennie is described to be â€Å"puzzled† and thathe â€Å"giggled happily† at some parts of their conversation, showing that he is unaware and immature. He constantly forgets everything very easily and doesn’t sense the seriousness in some of their conversations—showing that his maturity is like of a 6 year-old, who needs constant reminding and explaining of almost every matter discussed. Steinbeck states that after Lennie created ripples in the water with his fingers, he said â€Å"Look George, look what I done. He is easily impressed at the ripples he made and notifies George of his work to make him proud. Lennie’s child-like character also depicts a father/son relationship between Lennie and George. Earlier in the book, it is stated that â€Å"Lennie’s closed hand slowly obeyed† after George had commanded Lennie to hand over a dead mouse. The adverb â€Å"slowly† shows that though Lennie was reluctant to follow George’s orders, he had no choice but to obey him. Like an obedient son who must follow his father, Lennie fears George—the paternal figure—conveying George’s authority over Lennie. Supporting this, Lennie’s timid behaviour towards George is constantly portrayed as he is described to be speaking â€Å"slowly† and â€Å"cautiously† to George—indicating Lennie’s high level of respect for George. As a son would look up to his father, Lennie also sees George as his role model and a leader. Steinbeck portrays Lennie as submissive—giving him the role of the follower between the two main characters. Lennie is said to have â€Å"imitated George exactly† proving that he sees George as a role model. He â€Å"imitates† George, depicting his respect for him. Lennie also prioritizes him and whatever he says. This is seen when he tries to recall a memory from the past saying, â€Å"and you says†¦you says†. It is conveyed through this quote that he is dependent on George as he values George’s opinions more than his opinions. Supporting the fact that Lennie is dependent on George, George says, â€Å"think I’d let you carry your own work card? † This proves that George is much more responsible than Lennie, who constantly forgets things. Steinbeck also states in the beginning of the book that they walked in a â€Å"single file†, conveying that one is the leader and the other is the follower. Their relationship evidently highlights George’s authority over Lennie.

How Does Advertisement Influence People’s Behaviour?

In the modern world, advertisement is everywhere. In every abundance walk of life, there are huge competitions. As a result, advertisement has become more important. If you can be more noticeable, it means you would have chances to market. Therefore, advertising has great impact on different people. Advertising, is mainly used in market, refer to marketing message, which is presented by an identified sponsor in extinctive media such as the television, newspapers, radio, magazines and Internet. The term may be used to refer to message presentation in the marketplace.The term may also be extended to show a product in a television program or movie, which in order to target audience. This research paper focus on how does advertisement influence children and women on the society? Nowadays, advertisements can be found everywhere in our daily life.When you are walking on the street, you can see the advertisements in the shops or markets. When you are watching TV, advertisements appear betwe en programs. When you are reading newspapers, there are advertisements in the newspapers.Since advertisements are so popular in our daily life, they have great impact on the society and people’s behaviors. The influence can be in positive and negative ways. First of all, since advertisements are what we use to provide information for the customers, through advertisements, customers can get a lot of useful information like the functions of a product, the price of it and where they can buy it. In a manner of speaking, advertisements give us suggestions on what to buy and how to improve our life quality. Secondly, good advertisements can always stimulate consumer’s purchase desire.Such advertisements can usually help increase the sales of a kind of product. Therefore, advertisements can boot the profit of a company and stimulate the economy of a district. And they may create new fashion trends. Thirdly, good advertisements are usually great works of art, too.They are beau tiful and creative. So they have positive impacts on both the society and the people. Through advertisements, social aesthetic level can be raised, and people will be encouraged to appreciate beauty and to be creative. However, advertisements are not all good.They have bad impacts on the society and people’s behavior as well. As we all know, advertising is the most commonly used way by the companies to increase the sales. As a result, companies will try their best to attract audience even some times neglect bad impacts.Advertising is routinely being targeted to children in all over the world. â€Å" Media’s influence on children is mainly due to two dimensions-advertising and editorial/programming contended(O’ Guinn and Shrum,1997) –with advertising specifically intended to inform young consumers about products and encourage their purchase.†1 Because advertising makes effect on children, the media must impact the children, in other words, the child ren must reach the media, and then, interact with them. Children play passive role in the learning process, and they are good at imitating. When they are growing, they are trying to find a person such as their parents as models.Some of them will study on magazines; watch movies and TV shows to find out how to be a successful boy or girl. From the most fashionable hairstyle, the most popular music and dance, to the views to different kind of things.There are two ways that children can obtain about products. One is from parents, and the other one is from peers. Parents as consumer socialization agents. In western nations, it has been shown that parents teach children how to choose products, which are satisfied in the marketplace.Meanwhile, teaching them understand how to compare products in price and quality. Evidence shows that the more often that parents take young people shopping, the more aware the young people turn to purchasing information. Moreover, the communications among fam ily members may also impact young people’s interactions.Another significant evidence suggests that advertisement in television effect children a lot. Media influences most children’s requests for advertised products. Another important way young people can be affected is peers. At school, children spend long time studying or playing together.They can learn from each other what clothing styles are popular right now or how to wear can get more attention from others. In addition, peers influence a lot in children’s interaction for products. For example, they learn from their friends which stores are cheaper, which brands of selected products are much better.It is logical that if child A and child B are good friends, they always paly together, and if child A likes a brand of products, then child B will be influenced by child A, and start loving this brand of products. Also, there is an increasing evidence show that the more children affect each other, the more produc ts will be purchased by children.Thus, children are the huge consumer group in the world. The effect of advertisements on women in society. In the history, there is a standard of â€Å"beauty† during every period of time.But never like today, all kinds of media, especially television media, trying to teach us what is beauty should look like. In order to satisfy the needs males, advertisements are trying to define female beauty. Try to make women be seen as the aesthetic object and use this kind of â€Å" copy beauty† to attract consumers.Meanwhile, as a target group of consumer, women are apparently influenced by advertisements during watching process. The images in the advertising become emulating the target. In these pictures, the models are wearing pretty clothes, and they are having perfect bodies, long hair.They look sexy and attractive. In the society, this kind of advertising images are seen everywhere. Also, it influences women how they should look like and dre ss. For example, when several graceful air hostess who are dressing in a suit, having the long hair floats walking row tidy out from the plane.And telling others the reason why they have beautiful hair it is because they use â€Å"LOREAL† shampoo. After that, do you think women can just wait or ignore? All above, the unreal â€Å"female beauty† standard misled the female consumption. According to the stereotype image, there is few woman can achieve â€Å" young† and â€Å"beauty†.So, women start to re-create themselves. They use cosmetics, beauty, breast augmentation surgery and even cosmetic surgery to make themselves follow advertising images’ beautiful figures. As a result, women fall into the trap that shops made. Women accept advertising values, aesthetics propaganda and at the same time, they will act.Eventually, advertisement leads them to consume. It can be said that the whole industry are describing the â€Å" perfect female† in adv ertising, meanwhile, using women’s fear and upset feeling which is they are not as good as the models in the advertising to sell products to promote  consumption.Therefore, because of stereotyped advertisement, the consumption from women becomes modeled. â€Å"One study of a sample of Stanford graduates and undergraduates found that sixty-eight percent of students felt worse about their own appearances after reading women's magazines† (Crafting a Perfect Body).For example, â€Å"Allure† magazines, in 30 advertisements, 90% of them are thin Caucasian women who have long golden hair and blue eyes. Even the African American women, they changed hair to make it light and straight and try to look like Caucasian women.