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DNA sequencing is an analytical technique carried out in order to define the order of nucleotide bases in a DNA sample. The methods for carrying out this sequencing include the Maxam Gilbert Chemical Cleavage Method and the Sanger Chain Termination Method. The most commonly used method today is the Sanger method due to greater efficiency and less toxic materials used in the process compared to the chemical cleavage method.
DNA sequencing is important for a number of reasons. The sequence of nucleotide bases in DNA is vital in understanding human biology through identifying genes and other more specific fields including the identification and therapeutic treatment of genetic diseases.
In the past it is clear that extensive research has been carried out regarding the function of DNA and many people have contributed to research into the methods of DNA sequencing and its many uses.
A few of the many scientists and researchers who have contributed to DNA Sequencing over the last fifty years include Allan Maxam, Walter Gilbert, Frederick Sanger and Alan Coulson.3 The work carried out by these scientists laid the foundation for DNA sequencing methods used today.
Sanger and Coulson developed the plus-minus method in 1975 which was to be the first process used to sequence nucleotide bases; however, it could only be applied to a single stranded sample of DNA. This method was soon dwarfed by the work of Maxam and Gilbert who developed the chemical cleavage method just two years after the plus minus method. This method was favoured as it was capable of determining the sequence of bases in double stranded DNA helices. This work did not herald the end of research into the methods of DNA sequencing however, not long after Maxam and Gilbert published their work in 1977, the Sanger chain termination method was born. This method could be applied to double stranded DNA and was shown to be much more efficient and used a great deal less toxic chemicals.4 Thus, the Sanger method is commonly used today, however, novel techniques for sequencing DNA have been developed and offer significant advantages over the Sanger method. These techniques include: The 454 Genome Sequencer, Illumina/Solexa Genome Analyzer, SOLID System, and Helicos single-molecule sequencing device.
Autoradiography ¿½ Radioactive Labelled Tracer
Radioactive tracers are extremely powerful in molecular biology because of the really miniscule amounts of substances we are trying to detect and measure and the great sensitivity of the equipment used to measure radioactivity. In contrast, if we were trying to measure such tiny amounts using UV light absorption or by staining dyes, measurement would not be possible because of the limited sensitivity of these methods.
One of the most popular methods used to detect radioactivity by molecular biologists is autoradiography. This is done using a photographic emulsion which is normally a piece of x-ray film. After a sample of DNA has undergone electrophoresis on a gel, then x-ray film is then placed in contact with the gel and left in a dark room for a few hours or perhaps days if the radioactivity is weak. Exposure to the film is caused by the radioactive emission from the DNA, in the same way as light would. Thus, a film is produced and we see dark bands corresponding to the DNA bands on the gel. This is called autoradiography as the DNA bands take a picture of itself.
Automated Fluorescence Sequencing ¿½ Dye Based Sequencing
Even though there are enormous advantages of radioactive tracers with regards sensitivity, there is a significant disadvantage due to the potential health hazard and also the problem of radioactive waste once analysis is complete. But now non radioactive tracers can rival their sensitivity. In 1986, Leroy Hood and colleagues set developed a DNA sequencing method which replaced radioactive labelling, autoradiography and manual base calling with fluorescent labelling, laser induced fluorescence detection and computerised base calling. This method announced the age of automated, high throughput DNA sequencing. This method is a technical variation of the chain-terminator method (Sanger Method), where the primer is labeled with one of four different fluorescent dyes, and each dye marked primer is placed in a separate sequencing reaction containing one of the four dideoxynucleotides and all four deoxynucleotides.When the reactions are complete, they undergo electrophoresis and a fluorescence detector scan the gel as the reaction products migrate past. The fluorescence signature of each fragment is sent to a computer where software performs the base calling. Going one step further, Du Pont improved on this method by labelling the terminators themselves with different dyes. The main advantage of this is that a single reaction is necessary rather than four. As a result terminator dye sequencing is the mainstay in automated sequencing.
Maxam-Gilbert Sequencing (Chemical Cleavage Method)
Maxam-Gilbert DNA sequencing method allows us to break an end labelled DNA strand at a specific base using base specific regents. The method takes advantage of a two step catalytic process involving piperdine and two chemicals which selectively attack purines and pryrimidines. The first step is that the purines will react with dimethyl sulphate and pyrimidines will react with hydrazine in such a way as to break the glycosidic bond between the ribose sugar and the base. The second step, is that piperdine will then catalyze the phosphodiester bond cleavage where the bas has been displaced. The labelled substrate needs to be subjected to four separate cleavage reactions, each for the four different bases. As the strand is broken at each of repetition of that base (on separate strands of DNA), the lengths of the labelled fragments then identify the position of that base. The products or fragments of these reactions are resolved by size, by using electrophoresis in a high resolution polyacrylamide gel, and the DNA sequence can be deduced from the pattern of radioactive bands via autoradiography.
This method commences with a full length, end labelled DNA molecule; this can be at 3¿½ or 5¿½ end of the DNA molecule. Then, we modify a selected base. In the case of guanine, dimethyl sulphate is the reagent chosen which methylates guanine. Methylation is performed under mild conditions, as we do not want cleavage at every guanine base or else we will end up with tiny fragments that will not allow us to determine the sequence. Methylation under mild conditions allow on average only one methylated guanine base per DNA molecule. On addition of the reagent piperdine, the methylated base is lost and then proceeds to break the DNA backbone, at the site where the guanine base was lost. Thus a population of labeled fragments is generated, from the radio labeled end to the last "cut" site in the DNA strand. In order to be able to determine the whole DNA sequence it is necessary to run three other reactions that cleave at the other three bases. Dimethy sulphate and piperdine in formic acid will cleave both guanine and adenine bases. If we electrophorese this adenine and guanine reaction beside guanine only reaction, we can easily identify the adenine by comparison. Similarly, hydrazine and piperdine will cleave both cytosine and thymine bases, but in the presence of 2 M NaCl, hydrazine is specific for cytosine only, therefore we can run this reaction beside cystosine and thymine reaction and obtain thymine by comparison. Thus overall, we can deduce the sequence of the strand of DNA. In principle, we could just run three reactions, each of a single base specificity, using the absence of a band to identify the fourth base. This would be a non redundant method in which every bit of information was required. Such an approach may be subject to significant error and may lead to misinterpretation of a base. Redundant information serves as a check on the identifications.
Procedure To Perform Maxam-Gilbert Sequencing
* The DNA fragment (double strand) to be sequenced is end labelled by the addition of 32p at either the 3¿½ or 5¿½ end.
* The end labelled fragment is now cleaved with a restriction enzyme to produce two single strand enzymes or another method is to denature the double strand and separate the two strands on neutral polyacrylamide gel using electrophoresis.
* Four samples are exposed to the appropriate reagents and condition that cleave the relevant bases: To cleave guanine only, use dimethy sulphate followed by piperdine. To cleave adenine and guanine use dimethyl sulphate in formic acid followed by piperdine. To cleave cystosine and thymine use hydrazine followed by piperdine and to cleave cytosine only, use hydrazine in 2M NaCl followed by piperdine.
* The reaction proceed long enough to produce one break per strand, the random breaks generate end-labelled fragments representing all positions of each base.
* The sequence can easily be read from the pattern of bands after autoradiography.
Oligonucleotides are generally short nucleotides (nucleic acid polymers) and are approximately 20 bases in length. Larger oligonucleotides ranging from 160 to 200 bases are possible with automated synthesisers however due to the errors that can accumulate as the length of the oligonucleotide grows, 15 ¿½ 25 bases is the norm. Oligonucleotides composed of DNA are critically important in the polymerase chain reaction (PCR) replication process. In order to initiate DNA synthesis there must be a short double-stranded molecule region to provide a 3¿½ end onto which enzymes can add new nucleotides. The oligonucleotide acts as a primer in order to initiate the synthesis of a new polynucleotide. The base sequence of the oligonucleotide determines the position at which it attaches to the template DNA and hence specifies the region of the template that will be copied. The benefit of the oligonucleotide is that the position within the template molecule at which DNA copying is initiated can be specified by synthesizing a primer with the appropriate nucleotide sequence. A short specific segment of a much longer template molecule can therefore be copied, which is much more valuable than the random copying that would occur if DNA synthesis did not need to be primed (RNA synthesis does not require a primer). Oligonucleotides synthesis occurs by the chemical synthesis (polymerising) of individual nucleotide precursors. In the case of DNA synthesis, the polynucleotides being synthesised are in the 5 -> 3 direction, however chemical oligonucleotide synthesis is carried out in the opposite 3 -> 5 direction. The chemical process is described in detail by
Polymerase chain reaction (PCR)
Polymerase chain reaction application is particularly common in modern microbiology and forensic science. The characteristic of PCR is unique compared to the other scientific method in DNA sequencing. Polymerase chain reaction only requires a small amount of DNA information to amplify DNA sequences. There are many advantages of the PCR application process. This reaction can generate efficient laboratory diagnostic test in forensic science and it can also manipulate gene genome in DNA profiling to create clones DNA. The principle of PCR is to amplify two oligonucleotide primers in the 17-30 position in nucleotides chain. Hybridization of the 5¿½ and 3¿½ primers activate DNA replication. The following reaction involves the oligonulceotides to initiate DNA synthesis as previously stated.
The initiate stage of polymerase chain reaction is mainly dependant on the effect of heat to denature DNA polymerase. The thermal effect will alienate hydrogen from the nucleotide to fabricate fragments of DNA. Then the small segment of DNA will undergo cooling effect for the annealing of 3¿½ and 5¿½ primers. This process will lead to the development of DNA synthesis with the DNA base template. The repeated ongoing heating and cooling effect in PCR is important for the building of long stranded of DNA. In PCR technique a special type of thermophilic bacterium named Thermus Aquaticus that lives in hot spring that is essential for the amplification of DNA. The enzyme released from the thermophilic bacterium allows the protein within segment of the DNA to tolerate high temperature. The extreme temperature ranging from 75 to 90 degree Celsius is the optimal environment required for DNA replication in PCR. Once all the procedure had met as previously mentioned. DNA synthesis will occur and synthesising the complementary to the DNA template to create extension of DNA strand. DNA extension engages the 5¿½ and 3¿½ primers linkages process to the phosphate and hydroxyl group to generate long strand of DNA. The resultant process will lead to exponential amplification of small segment of DNA. Exponential growth of fragmented DNA can direct replication of millions copies of DNA strands. Therefore only a small sample of DNA is needed to trace back to the original source.
The term of Pyrosequencing¿½ is a registered trademark of Biotage AB which is a subsidiary of QIAGEN Corporation. Pyrosequencing¿½ technique offers an alternative to the well known chain termination method (Sangar method) for DNA sequencing. The pyrosequencing¿½ method can be used for determining the single nucleotide polymorphism analysis, tag sequencing with many DNA base pairs and the whole genome sequencing. The hypothesis of pyrosequencing¿½ is based on DNA sequencing by synthesis. This application involves in producing a single stranded DNA from a sequencing primer. The single stranded DNA act as a template for the enzymatic synthesis to occur within the complementary strand. In order for this process to happen, enzymes and substrates must be present. These enzymatic substances are Apyrase, ATP Sulfurylase, Luciferase and DNA polymerase. The substrates are Luciferin and Adenosine 5¿½ Phosphosulfate. The enzyme DNA polymerase has an important role in the transformation of Deoxyribo-Nucleotide Triphosphate into the DNA strand. The integration of (dNTP) involves in binding the correct DNA complementary to the template strand. Each successful combination of the base pairs into DNA strand will follow by the release of pyrophosphate (PPi). The (PPi) released is equal to the amount of merged nucleotides. Therefore, Pyrosequencing is highly dependent on monitoring the release of pyrophosphate from nucleotides bindings. Pyrophosphate will undergo conversion into ATP which is then gives off light to be detected by a charge coupled device chip. The resultant of ATP light signal output determines the signal data in the pyrogram trace. Hence, the pyrogram trace represents the light signal generated from the pyrophosphate which is proportional to the merged nucleotide in DNA sequencing.
DNA Sequencing ¿½ Chain Termination Method
The chain method, also called the Sanger method and dideoxy DNA sequencing, was discovered by Fredrick Sanger and his co workers in 1977. At first it was less popular than Maxam-Gilbert method, but after some improvements to the procedure it quickly took over as the method of choice. The advantages it holds over the Maxam-Gilbert method are that it is less technically complex, uses less hazardous chemicals, and is easier to scale up and can be fully automated . The chain termination method can allow for the reading of up to 1,000 bases of DNA per second .
The key to this sequencing approach is the use of dideoxynucleotides (ddNTPs). These molecules are the same as deoxynucleotides, with one important difference ¿½ there is no hydroxyl group at the 3¿½ carbon position. This means that DNA polymerase cannot add further nucleotides after a ddNTP in a DNA strand, and thus the strand is terminated at a defined point.
The modern Sanger method involves 4 distinct steps;
1. DNA preparation
2. Sequencing reaction
3. Capillary electrophoresis
4. Computer analysis
1. DNA preparation
The DNA must first be extracted from the cells of interest. This is done by breaking down the cell walls, using mechanical methods (grinding, blending, sonication) or chemical methods (detergents, solvents, enzymes). The DNA is then released and can be precipitated from the lysate, usually using an alcohol (ethanol or isopropanol typically).
The isolated DNA is then broken up into more manageable segments. The segments of DNA are inserted into plasmids, and these vectors are placed into a host bacterium. The bacteria containing the target DNA are placed into a culture media and allowed to multiply a million fold or more. This results in the target DNA being multiplied or cloned millions of times.
2. Sequencing Reaction
A polymerase chain reaction is used to maximise the number of replicated strands, of different lengths (due to the random uptake of ddNTPs), of the DNA under investigation. There are four main steps in the sequencing reaction;
1. First the cloned DNA fragments are heated up to disrupt the hydrogen bonds holding them together, separating them into single strands.
2. A small section of DNA, maybe 20 bases in length, called an oligonucleotide, is annealed to the template DNA strand. This short section of DNA is called a primer, and is required for the enzyme DNA polymerase to begin replicating a new DNA strand. It is present in excess, and with the help of rapid cooling anneals to the template DNA before the complementary DNA strand can reattach. The oligonucleotide primer must have a complementary sequence of bases to the template strand of DNA in order to attach, and this ensures that the primers attach at the same point in the DNA every time.
3. DNA polymerase then begins constructing a new DNA strand from dNTPs contained in the reaction mixture. The new dNTPs are added according to the complementary base pairs on the template strand, beginning at the primer and moving along the template. This process is called extension.
4. This continues until a dideoxynucleotide is added to the new strand by DNA polymerase, and chain termination occurs. When this happens the strand stops growing, as ddNTP lacks the hydroxyl group at the 3¿½ carbon position required to add another dNTP.
This whole process is then repeated many times, with the DNA being heated up again to split the strands, and then cooled rapidly to allow the primer to anneal to the template strand before the complementary DNA strand. This is called thermal cycling.
The concentration of ddNTPs is such that they are only taken up by DNA polymerase occasionally and termination events do not occur often. Each of the four ddNTPs carries a different fluorophore label. A fluorophore is a chemical group which fluoresces, and this can be read using an optical system. In the end DNA strands that have been stopped at every possible base in the sequence are obtained, each labelled according to the base at which extension was terminated by a ddNTP.
Below is a table summarising all of the components of the DNA sequencing mixture discussed in the method above, along with their function. An idea is given as to the concentration of the different components, relative to other components in the mixture, or the mixture as a whole.
3. Capillary Electrophoresis
Now that a population of DNA strands which have been terminated by a ddNTP at every possible base in the DNA sequence has been obtained, the strands must be organised according to length.
The DNA strands are first denatured again, using heat to separate the newly synthesised strands from the template DNA. The new strands are then loaded onto a very thin, long glass capillary tube filled with a denaturing polyacrylamide gel. This gel allows the smaller fragments of DNA to travel through it faster than the longer fragments. It also contains a high concentration of a denaturing agent which will ensure that the DNA remains single stranded.
An electrical current is used to pull the negatively charged DNA strands through the gel. Capillary electrophoresis can achieve a resolution of one base, and so DNA fragments that differ in length by even a single nucleotide can be size-fractionated . As the DNA strands come out of the bottom of the capillary, they pass through a laser which causes the dye label attached to the ddNTP to fluoresce at a certain wavelength. This is picked up by a photocell which passes the information onto a computer.
4. Computer Analysis
The signal received from the photocell is displayed by the computer as an electropherogram. The sequencer program on the computer translates this tracing of signal from the photocell into the sequence of nucleotides and prints this information across the top of the electropherogram plot. As the successive peaks of the electropherogram correspond to DNA segments differing in length by one nucleotide, the sequence of peaks corresponds to the sequence of bases in the DNA .
The next Generation
The demand for high-throughput, low cost sequencing has driven the development of next generation sequencing technologies. New applications in biology and medicine are becoming a reality taking things far beyond the original scope of genomic sequencing. Up until the human genome project the industry relied heavily on the Sanger-sequencing method, however when the international community decided that the whole genome sequence need to be determined it sparked the development of new techniques allowing higher sequencing throughput. Examples of new applications include ref  personal genomics, precision analysis for gene expression, analysis of functional regulation of gene expression, genomic-wide characterisation, profiling mRNAs, small RNAs, transcription factor regions, structure of chromatin, DNA methylation patterns, microbiology and metagenomics.
1.0 The 454 GenomeSequencer
454 GenomeSequencing was the first in a new breed of DNA sequencing platforms that produced high throughout sequencing. It is based on the principle of sequencing-by-synthesis and uses emulsion PCR of DNA library fragments affixed to micro-beads.
DNA fragments are ligated with specific adapters that cause the binding of one fragment to a bead. Emulsion PCR is carried out for fragment amplification with water droplets containing one bead and PCR reagents immersed in oil. The amplified DNA molecule inside the water droplets contains a single DNA template attached to a single primer-coated bead that then forms a clone clonally. The amplification is necessary for reliable detection in the reaction steps. When PCR amplification cycles are completed and after denaturation, each bead with its one amplified fragment is placed at the top end of an etched fibre in an optical fibre chip. Each bead sits on an addressable position in the light guide chip, containing several hundred thousand fibres with attached beads. The polymerase enzyme and primer are added to the beads, and one unlabelled nucleotide is supplied to the reaction mixture to all beads on the chip so that synthesis of the complementary strand can start. Incorporation of a following base by the polymerase enzyme in the growing chain releases a pryrophosphate group, which can be detected as emitted light. Knowing the identity of the nucleotide supplied in each step, the presence of a light signal indicates the next base incorporated into the sequence of the growing DNA strand. Of all next-generation platforms 454 sequencing provides the longest sequence reads, making it well suited to de novo genome assemblies. However, a drawback of the system¿½s chemistry is inaccuracies in calling homopolymeric stretches of sequence(i.e. AAAAA, CCCCC).
2.0 Illumina/Solexa Genome Analyzer
The principle is similar to the 454 platform in the parallel sequencing of DNA however instead of using agarose beads for amplification, clonal DNA clusters are generated by bridge amplification on a glass surface which facilitates increased densities of DNAs to be monitored simultaneously. The analyzer uses reverse terminator chemistry to sequence up to 100 million clonal DNA clusters in parallel. This mode of sequencing overcomes problems in quantifying the number of bases present in homopolymer stretches that is intrinsic to pyrosequencing. The sequence read length achieved in the repetitive reactions is about 35 nucleotides. A sequence of at least 40 million polonies can be simultaneously determined in parallel, resulting in a very high sequence throughput, of the order of Gigabases per support.
3.0 SOLID (Applied Biosystems) system
In a similar fashion to 454 pyrosequencing, DNA libraries are amplified on beads by emulsion PCR and the clonal sequence represented on each bead is determined by sequential rounds of ligation to a collection of dinucleotide-encoded adapters. The SOLID achieves high sequence accuracy because each base is interrogated twice in sequential rounds of ligation to dinucleotide-encoded adapters; once as the first base of the dinucleotide, and again as the second base of the dinucleotide. This re-reading of sequence minimises base-calling errors and makes the SOLID well suited to high-accuracy sampling applications.
4.0 Helicos single-molecule sequencing device
Helicos introduced the first commercial single-molecule DNA sequencing system in 2007. The nucleic acid fragments are hybridised to primers covalently anchored in random positions on a glass cover slip in a flow cell. The primer, polymerase enzyme and labelled nucleotides are added to the glass support. The next base incorporated into the synthesised strand is determined by analysis of the emitted light signal, in the sequencing-by-synthesis technique. This system also analyses many millions of single DNA fragments simultaneously, resulting in sequence throughput in the Gigabase range.
5.0 Novel DNA sequencing techniques in development
Multiplex polony technology facilitates several hundred sequencing templates are deposited onto thin agarose layers, and sequences are determined in parallel. An increase of several orders of magnitudes in the number of samples can be analysed simultaneously. Another benefit is the large reduction of reaction volumes, the smaller amounts of reagents needed and the resulting lower cost.
Real-time single-molecule DNA sequencing is produced using a specially engineered DNA polymerase with a donor fluorescent dye incorporated close to the active site involved in selection of the nucleotides during synthesis. All four nucleotides to be integrated have been modified, each with a different acceptor dye. During the synthesis, when the correct nucleotide is found, selected and enters the active site of the enzyme, the donor dye label in the polymerase comes into close proximity with the acceptor dye on the nucleotides and energy is transferred from donor to acceptor dye giving rise to a fluorescent resonant energytransfer (FRET) light signal. The frequency of this signal varies depending on the label incorporated in the nucleotides, so that by recording frequencies of emitted FRET signals it will be possible to determine base sequences, at the speed at which the polymerase can integrate the nucleotides during the synthesis process (usually a few hundred per second). The acceptor fluorophore is removed during nucleotide incorporation, which ensures that there are no DNA modifications that might slow down the polymerase during synthesis.
Next generation sequencing has been applied to the re-sequencing of previously published reference strains and allowed for the identification of all mutations in an organism at the genomic level. Next generation sequencing will allow for direct access to deciphering the cells transcripts on the sequence level and promises, at a much lower sequencing cost, the ability to assess plant genomes such as the 16-Gb hexaploid genome in wheat. In the medical field, applications include cancer genetics such as detection of specific cancer alleles through ultra-deep sequencing of genomic DNA. Other applications include metagenomics analysis with the field of ancient DNA research such as deciphering the Neanderthal genome.