Heat-stable polymerase

Section 4.3), which had to be replenished at every new cycle, the application of heat-stable polymerases from organisms such as Thermus aquations (Taq polymerase), Pyrococcus woesii (Pwo polymerase), Pyrococcus juriosus (Pfu polymerase), or Thermococcus litoralis (Vent polymerase) facilitated automation of the thermal cycling process. 

Figure 4.2 Polymerase chain reaction (PCR) (Lottspeich, 1998). The PCR cycles between a denaturing step to obtain single-stranded DNA, an annealing step for the primer attachment to the template DNA, and a polymerization step, in which the heat-stable polymerase elongates the corresponding strand using the primers as starting points.

Immunocapture-polymerase chain reaction (IC-PCR) is a synthesis of two commonly used diagnostic tools. This method exploits the high-affinity binding of antibodies to provide a facile method of purification, usually from a complex matrix, supplying the substrate for PCR detection. PCR exponentially amplifies a deoxyribonucleic acid (DNA) template in a temperature-dependent fashion by the annealing of oligonucleotide primers, enzymatic extension of bound primers by a heat-stable polymerase, followed by denaturation of... 

DNA synthesis. The solution is then heated to 72°C, the optimal temperature for Taq DNA polymerase. This heat-stable polymerase comes from T hermus aq uaticus, a thermophilic bacterium that lives in hot springs. The polymerase elongates both primers in the direction of the target sequence because DNA synthesis is in the 5 -to-3 direction. DNA synthesis takes place on both strands but extends beyond the target sequence. 

A heat-stable polymerase (Taq) is usually used, which remains active through many heating and cooling cycles. 

The PCR process was first published by MuUis in 1985. It uses a mixture of the DNA to be duplicated, heat-stable polymerase, required by the polymerase, the four 2 -deoxyribonucleoside triphosphate building blocks (A,T,C,G), and the oligonucleotide primers (about 20 nucleotides long). There are three main steps in the process ... 

PGR amplification of a DNA sequence is faciHtated by the use of a heat-stable DNA polymerase, Taq polymerase (TM), derived from the thermostable bacterium Thermus aquaticus. The thermostable polymerase allows the repeated steps of strand separation, primer annealing, and DNA synthesis to be carried out ia a single reactioa mixture where the temperature is cycled automatically. Each cycle coasists of a high temperature step to deaature the template strands, a lower temperature annealing of the primer and template, and a higher temperature synthesis step. AH components of the reaction are present ia the same tube. 

FIGURE 13.21 Polymerase chain reaction (PCR). Oligonucleotides complementary to a given DNA sequence prime the synthesis of only that sequence. Heat-stable Taq DNA polymerase survives many cycles of heating. Theoretically, the amount of the specific primed sequence is doubled in each cycle. 

Extend DNA synthesis in presence of heat stable DNA polymerase at 72° C in presence of dNTPS. 

RNA amplification by PCR has been facilitated by the use of a single heat-stable enzyme. Thus, DNA polymerase from Thermus thermophilus, which has enhanced reverse transcriptase (rT) activity in presence of manganese, can be used with one buffer system. The high temperature used for rT (70°C) to produce a complementary DNA copy from RNA, and the subsequent amplification of DNA at 60°C, increases efficiency by destabilizing secondary structures in the RNA template. This procedure has been used for the amplification of hepatitis C viral RNA (Yl). 

PCR makes use of the heat-stable enzyme DNA polymerase from the bacterium Thermus aquaticus and its ability to synthesize complementary strands of DNA when supplied with the necessary deoxyribonu-cleoside triphosphates. We have already looked at the chemistry of DNA replication (see Section 14.2.2), and this process is exactly the same, though it is carried out in the laboratory and has been automated. 

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