DNA: Restriction Mapping and Nucleotide Sequencing

Read this article to learn about the restriction mapping of DNA which involves the size analysis of restriction fragments and also learn about nucleotide sequencing of DNA for which two techniques have been developed:

(1) based on enzymatic method called Sanger sequencing and (2) based on chemical method called Maxam and Gilbert sequencing.

Restriction Mapping of DNA Fragments:

This involves the size analysis of restriction fragments produced by several restriction enzymes individually and in combination. For example, in Fig. 3.2 restriction sites of two enzymes A and B are being mapped. Cleavage with A gives fragments 2 and 7 kilo bases from a 9 kb molecule, hence we can have position of single A site from one end.

Similarly, B gives fragments 3 kb and 6 kb away, so it has a single site 3 kb from one end; but it is still not clear whether this site is near A’s site or is at opposite end of DNA. This can be resolved by double digestion. If the resultant fragments are 2 kb, 3 kb and 4 kb away, then A and B cut at opposite ends of the molecule; if they are 1 kb, 2 kb and 6 kb away the sites are near to each other. It is worth stating here that the mapping of real molecules is rarely as simple as this.

Nucleotide Sequencing of DNA:

The precise usage of codons, information regarding mutations and polymorphisms and identifica­tion of gene regulatory control sequences can only be elucidated by analyzing DNA sequences. Two techniques have been developed for this, one based on an enzymic method frequently termed Sanger sequencing and chemical method called Maxam and Gilbert sequencing.

Sanger’s Sequencing or Dideoxynucelotide Chain Terminators:

In this the reaction mixture is divided into four groups, representing the four dNTPs A, C, G and T. In addition to all the dNTPs being present in the A tube; an analogue of dATP is added (2′, 3′ ddATP) is added that is similar to A but has no 3′ hydroxyl group and so will terminate the growing chain. Situation for other tubes ddC, ddG and ddT are identical except they contain ddCTP, ddGTP and ddTTp respectively.

Since the incorporation of ddNTP rather than dNTP is a random event, the reaction will produce new molecule varying widely in length, but all terminating at the same type of base. Thus four sets of DNA sequences are generated, each terminating at a different type of base, but all have a common 5′- end. The four labelled and chain-terminated samples are then denatured by heating and loaded next to each other for electrophoresis.

Electrophoresis is performed at 70°C in pres­ence of urea, to prevent renaturation of DNA. Very thin and long gels are used for maximum resolution over a wide range of fragment lengths. After electrophoresis, the position of radioactive DNA bands on the gel is determined by autoradiography.

Since every band in the track from dideoxyadenosine triphosphate must contain molecules that terminate at adenine, and those in ddCTP terminate at cytosine, etc., it is possible to read the sequence of the newly synthesized strand from autoradiograph, provided that the gel can resolve differences in length equal to single nucleotide. Under ideal conditions, sequences up to about 400 bases length can be read from one gel.

Direct PCR Sequencing:

It is possible to undertake nucleotide sequencing from double-stranded molecules such as plasmid cloning vectors and PCR products but the double-stranded DNA must be denatured prior to annealing with primer. In case of plasmids, an alkaline denaturation step is sufficient. However, for PCR products this is more problematic and a focus of much research. Unlike plasmids, PCR products are short and re-annealed rapidly, so preventing the re-annealing process or biasing the amplification towards one strand by using a primer ratio of 100:1 can overcome this problem to a certain extent.

Denaturants such as form amide or dimethylsulphooxide (DMSO) are usually em­ployed to prevent renaturation of PCR strands after their separation it is possible to physically separate and retain one PCR strands by incorporating a molecule such as biotin into one of the primer, which can be recover after PCR by affinity chromatography with streptavidin, leaving the complimentary PCR strand. Thus it provided high quality single stranded DNA for sequencing.

One of the most useful methods of sequencing PCR products is termed PCR cycle sequencing. This is not strictly a PCR, since it involves linear amplification with a single primer in about 20 PCR cycles. Radiolabeled or fluores­cent—labelled dideoxynucleotides are then introduced into the final stages of reaction to generate chain termination extension products. Automated direct PCR sequencing is increasingly being refined, allowing greater lengths of DNA to be analysed in one sequencing run.

Automated Fluorescent DNA Sequencing:

This involves dideoxynucleotides labelled with different flurochromes and are used to carry out chain termination as in standard re­actions. The advantage of this modification is that, since different labellel is incorporated in each ddNTP all the products are run on same denaturing electrophoresis gel.

Each product with their base specific dye is excited by a laser and dye then emits light at its characteristic wavelength. A diffraction grating separates the emissions, which are detected by charge couple device (CCD) and the sequence is interpreted by computer. In addition to real time sequencing, the length of the sequence that may be analysed is in excess of 500 bp.

Maxam and Gilbert Sequencing:

This is a chemical method of sequencing developed by Maxam and Gilbert and the method is often used for sequencing of small fragments of DNA such as oligonucleotides. A radioactive label is added to either the 3′ or the 5′ end of a double stranded DNA preparation. The strands are then separated by electrophoresis under denaturating conditions and analysed separately.

DNA labelled at one end is divided into four aliquots; each is treated with chemicals that act on specific bases by methylation or removal of bases. Conditions are chosen such that each molecule is modified at only one position along its length and every base in the DNA strand has equal chances of being modi­fied.

After modification reactions separate samples are cleaved by piperidine, which breaks phosphodiester bonds exclusively at the 5′-side of nucleotides whose base has been modified. The result is similar to that produced by Sanger method, since each sample now contains radiolabelled molecules of various length, all with one end common (the labelled end), and with the other end cut at the same type of base. Analysis of the reaction products by electrophoresis is as already de­scribed for Sanger method.