There are two main methods by which DNA sequencing is performed, the Maxam and Gilbert method, and the Sanger method. Both methods were developed in 1977 and initially they were used equally, however more recently preference has been given to the Sanger method. The Maxam and Gilbert method uses the principle of chemical degradation, and can be used on either double stranded or single stranded DNA. It requires the DNA fragment which is to be sequenced to be labelled at one end. This is done by adding a radioactive phosphate to the 3′- or 5′- end, or by adding a nucleotide to the 3′- end.
The next step involves base-specific cleavages, which occur in two parts. Initially the base is modified using specific chemicals, such as dimethyl sulphate to methylate at G bases, formic acid to attack purines A and G, and hydrazine to hydrolyse at pyrimidines C and T. Next piperidine is used to cleave the sugar phosphate backbone of the DNA at the site. Limiting incubations times or concentrations of components in the first step ensures a ladder of progressively longer molecules is created. The resulting four lanes on the sequencing gel – G, A+G, C+T, C – enables the sequence to be determined.
The Sanger method relies on the principle of enzymic chain termination and is the more common method for two main reasons. The most important is that it is more easily automated, and this is essential to speed up the process. The second is that the chemicals used in the Maxam method are toxic and therefore hazardous to the health of the researchers. The first step in the Sanger method is production of purified single-stranded DNA for use as a template. There are three main methods by which these can be obtained. One involves the use of the bacterial virus M13, as it is capable of producing single stranded DNA and also purifying it.
The DNA to be sequenced is cloned into the double stranded replicative form of M13 and then the engineered virus is used to infect E.Coli, enabling virus particles containing single stranded DNA to be manufactured in bulk. Normally the M13 vector used has been genetically engineered to contain a multiple cloning site (stretch of DNA with recognition sites for several restriction enzymes) into which the DNA to be sequenced can be inserted.
A second method to obtain a template is via DNA being cloned into a plasmid vector. The resulting DNA is double stranded and therefore must be denatured with alkali or by boiling. This is the most common method used to obtain a template because it provides two complementary single stranded DNA molecules and therefore both ends of the cloned DNA molecule can be sequenced. However, it is difficult to prepare plasmid DNA that is not contaminated with small quantities of bacterial DNA or RNA, and these can act as false templates or primers and disturb the reaction.
A third method for obtaining a template uses gel electrophoresis to separate the two strands in a length of double stranded DNA. This method relies on the principle that one strand is heavier than the other because it contains more purine nucleotides than the other strand which contains more pyrimidines. As a result of more recent advances in this field, it is now possible to completely avoid cloning of DNA into M13 or a plasmid vector, by using polymerase chain reaction (PCR) to generate segments of DNA. PCR products are linear double stranded lengths of DNA which can be directly sequenced into single strands.
The next step in the process of DNA sequencing via the Sanger method requires the following to be placed in a test tube together. The template, DNA polymerase, a short DNA sequence to act as a primer, the four usual deoxynucleoside triphosphates (dG, dA, dC, dT) and a small amount of one kind of dideoxynucleotide. DNA polymerases are enzymes that synthesise new polynucleotides complementary to a DNA template, and thus these are essential for the reaction to proceed. The discovery of thermalstable DNA polymerases, which led to the development of PCR, has also resulted in thermal cycle sequencing which has two advantages over traditional chain termination sequencing. These are that is uses double stranded rather than single stranded DNA as the starting material, and that very little template DNA is needed, so the DNA does not have to be cloned before being sequenced.
In traditional chain termination DNA sequencing, DNA polymerases can not begin synthesis on an entirely single stranded template, so synthesis must be prompted by attachment of a short, synthetic oligonucleotide. In DNA sequencing the primer is also used to specify the point on the template at which DNA synthesis should begin. The primer is also radioactively labelled to permit autoradiography at a later stage in the procedure. Deoxynucleotides must be present in the test tube because they are required to bind with the template in order to form a DNA sequence. Critical to the Sanger method are the dideoxynucleotides which differ slightly from their counterparts that occur naturally in DNA.
Dideoxynucleotides are so called because they lack not just the single hydroxyl group at the carbon-2 position in the sugar component, but also the hydroxyl group at the carbon-3 site. This means that in its triphosphate form a dideoxynucleotide can be added to a growing DNA chain, but can not form a phosphodiester bond with any other activated precursor. Therefore a dideoxynucleotide acts as a terminator at the site that it occupies, bringing DNA synthesis to a halt.
To enable an accurate DNA sequence to be obtained, the template is divided amongst four test tubes, each containing a small amount of one of the four dideoxynucleotides. Under the proper incubation conditions, the synthesis of new strands will begin in each test tube as nucleotides in their activated form are added to the primer. In each tube the dideoxynucleotide and its corresponding deoxynucleotide will be competing for incorporation into the molecule, however it is usually the deoxy form which will be incorporated as it is present in greater quantity. As a result each of the four reactions will generate a collection of DNA fragments of different sizes. The new strands will be radioactive owing to the label in the primer.
The next step in the process of DNA sequencing via the Sanger method is gel electrophoresis. Following denaturation, the products in each of the four test tubes are placed in four lanes side by side on the same gel and separated by gel electrophoresis. The chains are separated according to size, with the shortest pieces ending up closest to the bottom as they move fastest during electrophoresis. The radioactive label results in all the DNA chains being radioactive, and thus after running, the gel is dried and a sheet of photographic film is laid on top of the gel. Darkened bands are formed where the radioactive DNA is located, each corresponding to a piece of DNA of a particular length and revealing the position of a particular base. This process is known as radiography. The DNA sequence is ultimately obtained by reading off the sequences starting at the bottom and combining results from all four bases.
Advances are always being made in the development of automated DNA sequencing machines. A DNA sequenator has been designed which is able to sequence nucleotides at a rate at least ten times greater than that which can be achieved by more costly and time consuming manual methods. Another advance is the use of different flourophores in the four base-specific reactions, meaning that unlike conventional DNA sequencing, all four reactions can be loaded into a single lane.
The colour of each band is recorded by a computer that analyses the data and prints out the sequence. Originally sequencing of long pieces of DNA was done by cutting the DNA into smaller segments with restriction enzymes and then subcloning each fragment separately into M13. However nowadays a technique known as primer walking has been developed for such purposes. This involves initially sequencing the cloned DNA as far as possible using the primer belonging to the M13 or plasmid vector, and then using the newly obtained sequence to make another primer which is then sequenced, and then so on until the end of the cloned DNA is reached.
In conclusion it can therefore be seen that there are four main steps by which traditional DNA sequencing takes place. The first is obtaining a single stranded length of DNA, the second is combining the template, primer, DNA polymerase, deoxynucleotides and dideoxynucleotides in four test tubes and enabling the reaction to proceed. The third step is gel electrophoresis, and the final step is combining the bands to obtain the results. However, advances are always being made in order to decrease the time spent on DNA sequencing, and thus the procedure is becoming more and more automated.
Endnotes:
1. Instant notes in molecular biology by P.C. Turner, A.G. McLennan, A.D. Bates and M.R.H. White, pg 147
2. Genomes by T.A. Brown, pg 65
3. Understanding Genetics – A molecular approach by Norman V Rothwell, pg 490
4. Molecular Biology made simple and fun by David P Clark and Lonnie D Russell, pg 389