Polymerase chain reaction template addition

Figure 3.25 Polymerase chain reaction. The steps involved in the chain reaction are as follows (i) Incubation of the DNA at a temperature above 90 °C in order to separate the two strands of the DNA duplex, (ii) Cooling of the solution to about 50 °C to allow annealing of the primers to the template (i.e. the nucleotides bind to the template DNA according to the basepairing rules), (iii) Finally, addition of the polymerase and Mg ions to extend the nucleotide primer and complete the synthesis of the complementary DNA, which takes place at about 70 °C. (iv) The sequence (i) to (iii) is repeated to allow another extension to occur many repetitions can be carried out which results in enormous multiplication of the DNA strands. NTPs - deoxyri-bonucleoside triphosphates.

Fig. 1. Comparison of enzyme-linked immuno sorbent assay (ELISA, left) and immuno-polymerase chain reaction (IPCR, right). During ELISA, an antibody-enzyme conjugate is bound to the target antigen. The enzyme converts a substrate in solution to a detectable product. In IPCR, the antibody-enzyme conjugate is replaced by an antibody-DNA conjugate. The subsequent addition of a DNA polymerase enzyme (e.g., Taq), nucleotides and a specific primer pair uses the antibody-linked DNA marker sequence as a template for amplification of the DNA. The PCR product is finally detected as an indicator of the initial amount of antigen.

All ingredients are present in the reaction mixture, which is added to a lipid film, and liposomes are prepared containing all macromolecules (enzymes and DNA or RNA templates) as well as all substrate molecules (nucleotides, for example). Consequently, this procedure has to be performed very quickly otherwise, the enzymatic reaction would mainly occur outside the liposomes and a distinction between product molecules synthesized inside from those produced outside and entrapped later would be difficult to draw. After the formation of liposomes, the enzymes outside the liposomes have to be inhibited by potent inhibitors (inhibitors that do their job even in the presence of substantial amounts of phospholipids) or the liposomal dispersion has to be treated by digestive enzymes. This strategy has basically been applied in the case of the RNA replication by QP replicase inside oleic acid/oleate liposomes" and in the case of the polymerase chain reaction (PCR) inside POPC or POPC/PS liposomes." In the former case, EDTA was added after the formation of the liposomes to inhibit the non-entrapped enzymes (and the kinetics was followed after addition of the EDTA molecules), in the latter case, the non-entrapped DNA template molecules were digested by DNase I before the temperature was raised to 95°C and the polymerization started. 

Polymerase chain reaction (PCR) (Section 25.8) A method for multiplying (amphfying) the number of copies of a DNA molecule. The reaction uses DNA polymerase enzymes to attach additional nucleotides to a short oligonucleotide primer that is bound to a complementary strand of DNA called a template. The nucleotide that the polymerases attach are those that are complementary to the base in the adjacent position on the template strand. Each cycle doubles the amoimt of target DNA that existed prior to the reaction step, yielding an exponential increase in the amount of DNA over time. 

DNA polymerases catalyze the formation of polynucleotide chains through the addition of successive nucleotides derived from deoxynucleoside triphosphates. The polymerase reaction takes place only in the presence of an appropriate DNA template. Each incoming nucleoside triphosphate first forms an appropriate base pair with a base in this template. Only then does the DNA polymerase link the incoming base with the predecessor in the chain. Thus, DNA polymerases are template-directed enzymes. 

Fig. 6. DNA sequence analysis, (a) Simplified methodology for dideoxy sequencing. A primer, 5 -TCTA, hybridized to the template, is used to initiate synthesis by DNA polymerase, (b) Stmcture of 2, 3 -dideoxy CTP. When no 3 -OH functionaUty is available to support addition of another nucleotide to the growing chain, synthesis terminates once this residue is incorporated into the synthetic reaction, (c) Representation of a DNA sequencing gel and the sequence, read from bottom to the top of the gel, gives sequence information in the conventional 5 to 3 direction.

RNA and DNA polymerases catalyze the same reaction mechanistically, involving hydrolysis of a nucleotide triphosphate to release pyrophosphate and form a phosphodiester bond. In both cases, the order of nucleotide addition is specified by the template, and synthesis of the growing nucleic acid chain is in a 5 to 3 direction (the enzymes move in a 3 to 5 direction along the template strand). In addition to the obvious difference in substrates (RNA polymerase utilizes ribonucleotides, whereas DNA polymerase utilizes deoxyribonucleotides), these two enzymes differ in their requirements for initiating synthesis ... 

These enzymes copy DNA sequences by using one strand as a template. The reaction catalyzed by DNA polymerases is the addition of deoxyribonucleotides to a DNA chain by using dNTPs as substrates, as shown in Figure 8-7. 

DNA polymerases catalyze the step-by-step addition of deoxyribonucleotide units to a DNA chain (Figure 5 21). Importantly, the new DNA chain is assembled directly on a preexisting DNA template. The reaction catalyzed, in its simplest form, is ... 

Role of RNA intermediates (Herbert and Rich, 1999). The mutation rate of a genome is likely to increase when genetic information is passed through RNA whether RNA is a viral genome or a retrotransposon because RNA polymerase reaction is neither edited nor subject to post-replicative repair. In addition, hotspots of a genetic chain in RNA retrotransposons can result from nomandom patterns of a decreased fidelity strand transfer to other templates and untemplated extensions. 

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