Deoxyribonucleic acid (DNA) was first discovered and isolated by Friedrich Miescher in 1869, but it remained understudied for many decades because proteins, rather than DNA, were thought to hold the genetic blueprint to life. In 1953, James Watson and Francis Crick put forward their double-helix model of DNA, based on crystallized X-ray structures being studied by Rosalind Franklin. According to the model, DNA is composed of two strands of nucleotides coiled around each other, linked together by hydrogen bonds and running in opposite directions. Each strand is composed of four complementary nucleotides – adenine (A), cytosine (C), guanine (G) and thymine (T) – with an A on one strand always paired with T on the other, and C always paired with G. They proposed such a structure allowed each strand to be used to reconstruct the other, an idea central to the passing on of hereditary information between generations (Porreca, 2010).
Two methods for DNA sequencing were developed at the same time to reveal the order of nucleotides in a DNA fragment. The chemical degradation method of DNA sequencing involves base-specific cleavage of the DNA strands while the dideoxy chain termination method works by enzymatic termination of the growing strands. The dideoxy chain termination method can be carried out under mild conditions using enzymes and does not involve hazardous chemicals whereas the chemical degradation method involves toxic chemicals. Many advances were introduced in the dideoxy chain termination method to increase the speed and the accuracy of sequencing. Therefore, this method was widely used in high throughput genome sequencing technology (Smith, 1986).
DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases, adenine, guanine, cytosine, and thymine, in a strand of DNA .DNA sequencing may be used to determine the sequence of individual genes, larger genetic regions, full chromosomes or entire genomes, of any organism. DNA sequencing is also the most efficient way to sequence RNA or protein. In fact, DNA sequencing has become a key technology in many areas of biology and other sciences such as medicine, forensics, or anthropology (Sambrook & Russell, 2001).
Sequencing can be applied in a variety of field.
The success of a DNA sequencing protocol is dependent on the sample preparation. A successful DNA extraction will yield a sample with long, non-degraded strands of DNA which require further preparation according to the sequencing technology to be used. (Church, 2006).Original DNA sequencing methods
Techniques for sequencing DNA were originally developed in 1977 by two groups: Alan Maxam and Walter Gilbert, and Frederick Sanger. Sanger’s method is most commonly used. This method involves the synthesis of a new strand of DNA using the DNA to be sequenced as a template. The reaction begins when single strands of template DNA are mixed with primer (a short piece of DNA complementary to the 5? end of the region to be sequenced), DNA polymerase, the four deoxynucleoside triphosphates (dNTPs), and dideoxynucleoside triphosphates (ddNTPs). ddNTPs differ from dNTPs in that the 3? carbon lacks a hydroxyl group. In such a reaction mixture, DNA synthesis will proceed until a ddNTP, rather than a dNTP, is added to the growing chain. Without a 3?-OH group to attack the 5?-PO4 of the next dNTP to be incorporated, synthesis stops. Indeed, Sanger’s technique is frequently referred to as the chain-termination DNA sequencing method.
In order to obtain sequence information, four separate synthesis reactions must be prepared, one for each ddNTP. When each DNA synthesis reaction is stopped, a collection of DNA fragments of varying lengths has been generated. The reaction prepared with ddATP produces fragments ending with an A; those with ddTTP produce fragments with T termini, and so forth. If the DNA is to be manually sequenced, radioactive dNTPs are used and each reaction is electrophoresed in a separate lane on a polyacrylamide gel. The molecular weight of each fragment is determined by its length, so shorter fragments migrate faster than larger fragments. Because synthesis proceeds with the addition of a nucleotide to the 3?-OH of the primer (i.e., in the 5? to 3? direction) the ddNTPat the end of the shortest fragment is assigned as the 5? end of the DNA sequence, while the largest fragment is the 3? end. In this way, the DNA sequence is read directly from the gel from the smallest to the largest fragment.
DNA replication, Gel electrophoresis DNA is more often prepared for automated sequencing. Here the four reaction mixtures can be combined and loaded into a single lane of a gel because each ddNTP is labeled with a different colored fluorescent dye. These fragments are then electrophoresed on a polyacrylamide gel and a laser beam determines the order in which they exit the gel. A chromatogram is generated in which the amplitude of each spike represents the fluorescent intensity of each particular fragment. The corresponding DNA sequence is listed above the chromatogram. Fully automated capillary electrophoresis DNA analyzers are required for large projects. These sequencing machines are very fast and can run for 24 hours without operator attention. As many as 96 samples can be sequenced simultaneously, making it possible to sequence as many as 1 million bases per day, per sequencer. This level of automation, involving many sequencers running at the same time, is needed for the completion of whole-genome sequences (Sanger et al., 1977).
Maxam–Gilbert sequencing, referred to as the chemical degradation method, requires radioactive labeling at one 5? end of the DNA fragment to be sequenced (typically by a kinase reaction using gamma-32P ATP) and purification of the DNA. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). For example, the purines (A+G) are depurinated using formic acid, the guanines (and to some extent the adenines) are methylated by dimethyl sulfate, and the pyrimidines (C+T) are hydrolysed using hydrazine (Maxam & Gilbert, 1977).
The addition of salt (sodium chloride) to the hydrazine reaction inhibits the reaction of thymine for the C-only reaction. The modified DNAs may then be cleaved by hot piperidine; (CH2)5NH at the position of the modified base. The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus a series of labeled fragments is generated, from the radiolabeled end to the first cut site in each molecule. The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each showing the location of identical radiolabeled DNA molecules. From presence and absence of certain fragments the sequence may be inferred (Maxam & Gilbert, 1977).
This method, although based on very simple principles, came with a whole lot of trouble. First, it was time consuming. And that was supposing that everything went well on the first try. A lot of steps in the method could cause problems: the radioactive labeling process, the cleavage reactions, the gel set up, the electrophoresis, and the X-ray film developer. Using this method you could only confirm about 200–300 bases of DNA every few days. Maxam-Gilbert sequencing also required working with large amounts of radioactive material and working closely with hydrazine, which is a known neurotoxin. The development of other techniques, and the simplification of Sanger sequencing, caused chemical sequencing to lose its appeal. With the birth of next-generation sequencing, Maxam-Gilbert sequencing is almost extinct and many are claiming the same will happen to Sanger sequencing.
Figure 1: An example of the results of automated chain-termination DNA sequencing.
Advanced methods of DNA sequencing
Over the years the next generation of sequencing methods has been developed which are not based on the dideoxy chain termination principle. These methods are still under development and further research is still being done ( Porreca, 2010).
Large-scale sequencing often aims at sequencing very long DNA pieces, such as whole chromosomes, although large-scale sequencing can also be used to generate very large numbers of short sequences, such as found in phage display. For longer targets such as chromosomes, common approaches consist of cutting, with restriction enzymes, or shearing large DNA fragments into shorter DNA fragments. The fragmented DNA may then be cloned into a DNA vector and amplified in a bacterial host such as Escherichia coli. Short DNA fragments purified from individual bacterial colonies are individually sequenced and assembled electronically into one long, contiguous sequence. Studies have shown that adding a size selection step to collect DNA fragments of uniform size can improve sequencing efficiency and accuracy of the genome assembly. In these studies, automated sizing has proven to be more reproducible and precise than manual gel sizing.
Sequencing by hybridization
Sequencing by hybridization is a non-enzymatic method that uses a DNA microarray. A single pool of DNA whose sequence is to be determined is fluorescently labeled and hybridized to an array containing known sequences. Strong hybridization signals from a given spot on the array identify its sequence in the DNA being sequenced (Porreca, 2010).
This method of sequencing utilizes binding characteristics of a library of short single stranded DNA molecules (oligonucleotides), also called DNA probes, to reconstruct a target DNA sequence. Non-specific hybrids are removed by washing and the target DNA is eluted. Hybrids are re-arranged such that the DNA sequence can be reconstructed. The benefit of this sequencing type is its ability to capture a large number of targets with a homogenous coverage. A large number of chemicals and starting DNA is usually required. However, with the advent of solution-based hybridization, much less equipment and chemicals are necessary (Clyde, 2007).
Sequencing with mass spectrometry
Mass spectrometry may be used to determine DNA sequences. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has specifically been investigated as an alternative method to gel electrophoresis for visualizing DNA fragments. With this method, DNA fragments generated by chain-termination sequencing reactions are compared by mass rather than by size. The mass of each nucleotide is different from the others and this difference is detectable by mass spectrometry. Single-nucleotide mutations in a fragment can be more easily detected with MS than by gel electrophoresis alone. MALDI-TOF MS can more easily detect differences between RNA fragments, so researchers may indirectly sequence DNA with MS-based methods by converting it to RNA first (Blazewicz, 2004).
Microfluidic Sanger sequencing
In microfluidic Sanger sequencing the entire thermocycling amplification of DNA fragments as well as their separation by electrophoresis is done on a single glass wafer (approximately 10 cm in diameter) thus reducing the reagent usage as well as cost. In some instances researchers have shown that they can increase the throughput of conventional sequencing through the use of microchips. Research will still need to be done in order to make this use of technology effective (Chen et al., 2010).
This approach directly visualizes the sequence of DNA molecules using electron microscopy. The first identification of DNA base pairs within intact DNA molecules by enzymatically incorporating modified bases, which contain atoms of increased atomic number, direct visualization and identification of individually labeled bases within a synthetic 3,272 base-pair DNA molecule and a 7,249 base-pair viral genome has been demonstrated (Pareek et al., 2011).
Success of a DNA sequence
The success of any DNA sequencing protocol relies upon the DNA or RNA sample extraction and preparation from the biological material of interest.
A successful DNA extraction will yield a DNA sample with long, non-degraded strands.
A successful RNA extraction will yield a RNA sample that should be converted to complementary DNA (cDNA) using reverse transcriptase, a DNA polymerase that synthesizes a complementary DNA based on existing strands of RNA in a PCR-like manner. Complementary DNA can then be processed the same way as genomic DNA.
According to the sequencing technology to be used, the samples resulting from either the DNA or the RNA extraction require further preparation. For Sanger sequencing, either cloning procedures or PCR are required prior to sequencing. In the case of next-generation sequencing methods, library preparation is required before processing. Assessing the quality and quantity of nucleic acids both after extraction and after library preparation identifies degraded, fragmented, and low-purity samples and yields high-quality sequencing data.
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STUDENT NAME: BOKVELD AMAHLE
STUDENT NUMBER: 201403591
MODULE: MIC 513
TASK: ASSIGNMENT 1
TOPIC: METHODS OF DNA SEQUENCING