Biochemistry and Molecular Biology - Belyasova N.A. 2002

Fundamentals of Genetic Engineering
Construction and Analysis of Genomic Gene Libraries
DNA Sequencing

Determining The nucleotide sequence of DNA (sequencing) is one of the most important and often essential stages in genome research. Sequencing is typically performed on purified (homogeneous) single-stranded DNA fragments 100–200 NUCLEOTIDES in length. Such fragments can be obtained through the following Procedures: cloning is used to achieve homogeneity of recombinant DNA within a single clone; vector DNA is isolated from Cells; restriction Enzymes are used to cleave the cloned DNA segment into fragments of the specified size; and raising the Temperature induces DNA Denaturation to yield single-stranded molecules. A simpler method of DNA preparation (suitable for the Sanger method) involves cloning single-stranded DNA fragments into vectors based on the M13 phage.

Two sequencing Methods are widely used: the Maxam-Gilbert chemical Cleavage method and the Sanger chain-termination method. The Sanger chain-termination method is currently considered the most modern and less labor-intensive.

DNA Sequencing by the Chain-Termination Method. This method is based on The Use of specific DNA Synthesis terminators—2',3'-dideoxyribonucleoside triphosphates. These molecules are normally incorporated into growing DNA chains via their 5'-triphosphate groups, but they fail to form a phosphodiester bond with the subsequent deoxyribonucleoside triphosphate (dNTP). When a small amount of a given dideoxyNTP (e.g., ddATP) is introduced into the DNA synthesis reaction mixture alongside the four normal dNTPs, a set of chains specifically terminated by that ddNTP is synthesized. If four separate reactions are carried out with the same DNA fragment, each using a different ddNTP, a mixture of newly synthesized molecules of varying lengths will be produced in each reaction. Consequently, all DNA chains synthesized in such a system will share the same 5'-end (determined by the use of identical primers) and identical nucleotides at the 3'-end. These products can then be separated by gel Electrophoresis, and their nucleotide sequence can be determined from the relative lengths of the generated DNA chains (Fig. 20.9).

The sequence of steps in this method is as follows: 1) single-stranded DNA fragments, whose nucleotide sequence is to be determined, are obtained (DNA templates); 2) a set of ddNTPs is synthesized; 3) a short radiolabeled DNA fragment complementary to the 3'-end of the template is synthesized (primer); 4) four DNA synthesis reaction mixtures are prepared, each containing one specific ddNTP and the corresponding dNTP in a strictly defined ratio, along with the other three dNTPs; 5) DNA synthesis is allowed to proceed in each reaction mixture. If the ddNTP : dNTP ratio is properly chosen, a set of labeled molecules is formed whose lengths correspond to the distances from the given ddNTP residues to the beginning of the chain; 6) the resulting DNA mixtures are denatured to generate single-stranded molecules; 7) the fragments are separated by size using gel electrophoresis (molecules migrate through a porous agarose or polyacrylamide gel under an electric field at a speed inversely proportional to their size); 8) autoradiography is performed, and the nucleotide sequence is established from the distribution pattern of the labeled fragments.

The most modern Modification of the Sanger method involves the use of single-stranded DNA fragments cloned into M13 phage-based vectors. The insertion site for foreign DNA within the M13 genome is precisely known. Consequently, 8- to 10-mer oligonucleotides complementary to the M13 DNA region directly adjacent to the cloned sequence have been developed. These nucleotides serve as primers in polymerase reactions, thereby making it possible to use a single synthetic primer for sequencing any insert within that vector.

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Fig. 20.9. Scheme of DNA sequencing by the Sanger method

Under optimal conditions, this method allows researchers to determine The Structure of a fragment comprising hundreds of nucleotides in a single experiment.

DNA Sequencing by the Chemical Cleavage Method. To fix the starting point of DNA Restriction fragments, their 5'-ends are labeled with the radioactive phosphorus isotope ³²P using the enzyme polynucleotide kinase (Fig. 20.10). The DNA is then denatured to yield single-stranded chains, and the sample is divided into four portions. Each portion is treated with a reagent that specifically destroys one or two of the four possible nitrogenous bases. Reaction conditions are adjusted so that each DNA molecule sustains only a few modifications. Subsequently, the damaged molecules are treated with piperidine, which induces strand breaks at the nucleotides with damaged bases. This yields mixtures containing fragments of varying lengths, among which only the labeled ones have a "fixed" starting point (5'-end) and can be detected by autoradiographic analysis. For example, if guanylate was located at positions 3, 8, and 10 in a 10-nucleotide-long fragment, Treatment with a guanine-damaging reagent will generate chains of 3, 8, 10, 5, and 2 nucleotides. Of these, only the first three, as they contain the radioactive phosphorus isotope, will produce a dark band on the X-ray film.

Fig. 20.10. Scheme of DNA sequencing by the Maxam-Gilbert method

At The final stage, the sets of labeled fragments generated in each of the four reactions are subjected to electrophoresis in adjacent lanes of a polyacrylamide gel. The DNA fragments are separated according to their size. Gel autoradiography is then performed, and the nucleotide sequence of the analyzed chain is read. In this setup, the first nucleotide in the chain corresponds to the shortest DNA fragment, which appears last on the gel autoradiograph (Fig. 20.10).