| Chain termination (Sanger) DNA sequencing
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| Chain termination sequencing uses a DNA polymerase enzyme, a single-stranded template DNA and a sequencing primer
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| In this method, originally developed by Fred Sanger, the sequencing primer is designed to be complementary to the region flanking the sequence of interest and acts as the starting point of chain elongation. DNA polymerization requires dNTPs to allow the strand to be elongated but, in addition, radioactive chain-terminating dideoxynucleotides (ddNTPs) are added. These are analogs of the dNTPs but differ in that they lack the 3'-hydroxyl group required for formation of a covalent bond with the 5'-phosphate group of the incoming dNTP. Therefore, during DNA polymerization, if a growing DNA chain incorporates a ddNTP, growth of the nucleotide chain is halted. A total of four reactions are carried out in parallel, each containing the primer, template, polymerase and dNTPs. However, to each of the four reactions, a small amount of one radioactive ddNTP is added (ddATP, ddCTP, ddTTP, ddGTP) so that four separate reactions, the A, T, G, and C reactions, are conducted in parallel. As the chains elongate, ddNTPs, which are present at lower concentration than the natural dNTP, will be incorporated into the chain in place of the corresponding dNTP on a random basis. This means that in any one reaction mixture, there will be many chains of varying lengths, which, when pooled together, represent the total collection of fragments that could terminate at that base. The DNA chains of differing lengths can be separated by electrophoresis on denaturing polyacrylamide gels. These gels allow DNA fragments that differ by only one nucleotide in length to be separated and, if the reaction involves a labeled group, either a dNTP or the primer, then electrophoresis of the four reactions in parallel, with subsequent autoradiography, will allow the sequence of the DNA to be determined (Fig. 34.19). In general, this method can produce sequence data for the 300-500 bases downstream of the sequencing primer, but some protocols permit analysis of much longer sequences.
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| The chain termination method can be modified by replacing radioactive groups with fluorescence-labeled primers, which then allow sensitive monitoring of each DNA fragment as it reaches the bottom of the gel. This process lends itself to automation (Fig. 34.19).
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Figure 34.18 DGGE method to detect DNA mutations. (A) As double-stranded DNA migrates through a gel containing a gradient of increasing concentration of denaturants, e.g. urea breaks hydrogen bonds between the two strands. At a point unique to that particular DNA, all the hydrogen bonds will be broken and the two strands separate - the so-called melting point. If the nucleotide sequence of a DNA is changed, e.g. by a mutation, the melting point of the DNA will be altered and thus the mobility of the DNA in the gel will be altered, allowing the change to be detected by autoradiography. (B) DGGE with mutation. In this case, a single A > C substitution has occurred. This increases the number of hydrogen bonds between the two strands and alters the mobility in the denaturant gradient. The difference in mobility through the gel only highlights the difference in the two strands' nucleotide sequence - it does not say what or where the difference is. |
| REVERSE TRANSCRIPTASE-PCR |
| From whole blood, or pathologic tissue, cDNA for a gene can be prepared by the process of reverse transcription. Reverse transcription uses a reverse transcriptase enzyme to polymerize a DNA molecule complementary to the mRNA molecule, using a single poly T primer, which binds to the mRNA at its 3' poly A tail. The reverse transcriptase proceeds along the mRNA to manufacture a complementary strand, cDNA. The resulting cDNA is then used as a template for PCR where the entire cDNA can be amplified to produce a DNA molecule containing the entire coding sequence of a gene. Clearly in some cases, gene expression is tissue-specific and one would not expect some mRNAs to be present in blood, e.g. dystrophin from muscle. However, ectopic transcription of genes occurs in white blood cells at a low level and allows analysis of transcripts of genes not normally expressed. This can be useful if the desired transcript is derived from a tissue that is not easily accessible or if study of the cDNA can give definitive information about the presence of a mutation. |
| The cDNA can then be incorporated into a cell-based cloning vector for further amplification or can be introduced into a cellular expression system to produce the gene product in vitro. Such methods are used increasingly in the study of the functional aspects of mutant gene products. |
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| Figure 34.19 DNA sequencing using dideoxynucleotides. A single reaction involving the DNA template and primer will proceed until a ddNTP is incorporated into the chain at random. This halts the reaction and that particular chain can grow no longer. Four reactions are performed in parallel, and each reaction is identical other than the ddNTP that is included, ddATP, ddCTP, ddGTP, and ddNTP. Each reaction generates hundreds of different reaction products with the same 5'end, the sequencing primer, but differing 3' ends. The length of the 3' end will vary from 1 to over 300 bases depending on when a ddNTP was incorporated into the chain. In order to visualize the sequence a radiolabeled dNTP is added to all four reactions to facilitate autoradiography. Once completed, the four reactions are electrophoresized simultaneously on a polyacrylamide gel and the resultant autoradiograph is read and the sequence determined. |
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