6.9 Polymerase Chain Reactions in Analysis of the Primary Structure of Nucleic Acids

The enzymatic method of DNA sequencing has found broad application primarily because it can be combined with isolation and multiplication of fragments of the DNA under analysis by way of molecular cloning. It was noticed that these fragments are accidentally entrained by a vector from a complex mixture obtained when the polymeric DNA is cleaved in one or another way. In recent years, however, an effective procedure has been developed to produce strictly defined fragments of virtually any DNA, including the total human genome DNA. It is based on DNA-polymerase chain reactions (PCR) which immediately found extensive applications in different fields of molecular biology and gene engineering, including analysis of the primary structure of nucleic acids.

A PCR is based on the DNA polymerase-mediated buildup of the primer oligonucleotide on a single-stranded DNA template (Fig. 6-11). The potential of such a reaction has been drastically expanded by using in it Taq DNA polymerase isolated from the thermophilic bacteria Thermus aquaticus. The activity of this enzyme (recently, other enzymes of a similar type have been found as well) is at its peak at 70⁰ C and it can withstand heating to 95⁰C for a short period of time. By virtue of such properties it has become possible to copy and multiple any selected DNA fragment by means of Taq polymerase and two primers oriented toward each other, as is shown in Figure 6-33. The process boils down to running repeated cycles, including denaturation of the DNA at 90-94⁰ C, hybridization with primers at 50-60⁰ C, and polymerasemediated buildup at 70⁰ C. This gives rise to a chain reaction in which the newly synthesized DNA serves as a template for synthesis of yet another one during the next cycle. Thus, exponential multiplication of a DNA fragment takes place in the course of the PCR. The fragment can be multiplied by a factor of tens of millions over 25 to 30 cycles and produced in microgram quantities even if only a few copies were present in the starting DNA (which may be both double- and single-stranded). The length of the fragment may range anywhere from a hundred to several thousands of base pairs; the termini are determined by the nucleotide sequence of the primers or, in other words, can be defined in advance. The fact that primers of 20 to 25 units long (their synthesis has already become routine) can be used and the PCR can be conducted at an elevated temperature means that the reaction is highly specific and may practically yield a single product. The development of thermocyclers capable of changing the temperature of a large number of samples using a preset program has made it possible to fully automate such reactions.

It should be pointed out that the potential of PCRs is not limited to mere copying of selected DNA or RNA fragments (the latter is possible if a mixture of an RNA and two primers is first treated with reverse transcriptase, then a PCR is conducted to multiply the fragment of the resulting cDNA). PCR modifications have already been elaborated, whereby predetermined changes can be introduced into DNA and selected fragments of different DNAs can be interlinked, thus opening up greater possibilities for gene engineering.

The double-stranded DNA fragments yielded by the PCR can be easily sequenced by the enzymatic method already described. The primer oligonucleotide used in this case (it can, for example, be one of the PCR primers) can be readily hybridized with the corresponding strand of a duplex DNA. To this end, the DNA and primer mixture is heated to 90⁰ C then cooled rapidly, after which nucleoside triphosphates and DNA polymerase are immediately added. Sometimes, a single-stranded DNA is produced specifically for subsequent sequencing (as well as other purposes) in what has become known as asymmetrical PCR. In this case, two primers are introduced into the PCR in different quantities to give a mixture of a double-stranded DNA and a single-stranded product of primer build-up, taken in an excess amount. It should also be noted that the thermophilic DNA Taq polymerase can be used directly for sequencing as well. Then, the polymerase copying with termination is conducted at a higher temperature, which makes it possible to obviate the complications associated with the tendency of certain nucleotide sequences toward intramolecular complementary interactions.

By resorting to a PCR during sequencing of the genome DNA one can replace the time-consuming molecular cloning by a simple automated process. A particularly attractive feature of this reaction is its applicability to analysis of variable and mutant segments of the genome in comparative and evolutionary studies. By synthesizing primer pairs complementary with respect to the conservative regions confining such segments, one can analyze, within a single experiment, DNA preparations for a great number of individuals at a time. Moreover, analysis with a PCR provides access to DNAs in any form and in smallest amounts. PCR copying is also applicable to DNA in cells and tissues (e.g., hairs, preparations and smears from collections and herbaria, ancient and mummified samples), which opens up a brand new field of genetic research.

As can be seen from the general scheme of PCR (Fig. 6-33), in order to copy an unknown DNA fragment the latter must be preceded and followed by known sequences with which the primers are hybridized. In practice, however, it often becomes necessary to copy and sequence DNA fragments flanking a small known segment on either side. This can be done by means of an inverse PCR. In this case, the DNA under analysis is first cleaved with the aid of a restriction endonuclease, then the resulting fragments are cyclized using DNA ligase. Subsequently, two primers complementary to the known segment and oriented in two opposite directions toward unknown sequences are used for PCR copying of the resulting mixture. As a consequence, only the cyclized fragment containing the known sequence is linearized and multiplied. This sequence is arranged as two segments flanking the obtained product and can serve as a template for sequencing primer oligonucleotides as described above. Other modifications of the PCR have also been developed and are being used.

In conclusion, it should be pointed out that the PCR is ideally suited to automation of the sequencing procedure. By its nature this reaction already provides automation of most of the sample preparation process. As regards the automatic sequencers described below, they can handle PCR products just as successfully as those of molecular cloning.