Add thermostable DNA polymerase and deoxynucleotides

Incubate 70-75t for DNA synthesis by primer extension

; 111 n 111 n 111111111 n^m


Repeat cycle of denaturation, primer binding, DNA synthesis in automated theimocycler

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I I I I I I II I I I I I I I I I I I I I I I I I I I Repeat for approx. 30 cycles

Fig. 12-8 Amplification of part of a DNA sequence by the polymerase chain reaction (PCR) Oligonucleotide primers must first be made according to the sequences at either end of the portion of DNA to be amplified. After the DNA has been denatured by heating, the primers can hybridize to the complementary sequences on the opposite strand In the presence of heat-resistant DNA polymerase and deoxynucleotide triphosphates, two new copies of the desired region are produced. The cycles of melting, annealing, and extension arc repeated rapidly, each time, the amount of target DNA sequence doubles After the first few cycles virtually all the templates consist of just the short region chosen for amplification After 30 cycles, taking about 3 hours, the region bounded by the chosen primers has been amplified many millionfold (Courtesy Drs. I H. Holmes and R Strugnell )

can readily be identified using labeled probes in a hybridization assay. Moreover, the PCR can be modified for the detection of viral RNA, by incorporating a preliminary step in which reverse transcriptase is used to convert the RNA to DNA. It is not necessary or usual to amplify the whole genome, but it is necessary to know at least part of the nucleotide sequence, in order to synthesize two oligonucleotide primers representing the extremities of the region one chooses to amplify.

There are three steps in the process: (1) melting the target DNA at 95°C,

(2) cooling to 50C-60°C to allow binding of two oligonucleotide primers, corresponding with short sequences (on opposite strands of the template DNA) which flank the segment oi DNA that is to be amplified, and (3) extension from the oligonucleotide primers by DNA polymerase to form two DNA strands that are complementary copies of the original (plus and minus) target strands, that is, the DNA located between and including the two primers. The cycle of melting, primer binding, and primer extension is repeated many times, and the number of DNA copies increases exponentially. Thus after 30 cycles the number of DNA copies, beginning with a single copy of the target sequence, is over one million. Thirty cycles can be completed within 4 hours using a suitable automated thermal cycling device. By using the heat-stable DNA polymerase {Taq) of Thermus aquaticus, an organism naturally found in hot springs, it is not necessary to replenish the enzyme between cycles. The amplified DNA may be detected by agarose gel electrophoresis or by hybridization with labeled DNA or RNA probes culminating in any of a wide variety of readout systems (Fig. 12-9).

Selection of the most suitable pair of primers is a matter of central importance. They may be chosen to be highly specific for a particular virus strain or, alternatively, to represent consensus sequences within a gene that is conserved within a given family or genus. By probing for a conserved gene such as that for RNA polymerase it is even possible to discover a previously unknown agent. Reactions must be carried out under very carefully controlled conditions of ionic strength, temperature, primer concentration, and nucleotide concentration. Deviations can result in nonspecific amplification. The problem of contamination of samples with extraneous DNA has delayed the transition of PCR from research tool to routine diagnostic method, and great care is required to avoid such contamination. However, a number of technical refinements have been introduced, meticulous attention to which has made PCR a reliable methodology in experienced laboratories.

isothermal amplification is a new technique that does not require the temperature cycling and accompanying equipment of PCR. Three enzymes involved in the replication of retroviruses are simply mixed with either a DNA or RNA template and DNA primers at a constant temperature, and million-fold amplification is rapidly achieved.

These several developments have so dramatically enhanced the sensitivity and versatility of nucleic acid hybridization that probing for the viral genome may overtake probing for antigen as the diagnostic method of choice in many laboratories. The procedure is invaluable when dealing with (1) viruses that cannot be cultured satisfactorily, (2) specimens that contain predominantly inactivated virus, as a result of prolonged storage or transport, or

(3) latent infections in which the viral genome lies dormant and virus is not

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