Polymerase chain reaction primer synthesis

Oligonucleotide synthesis is used to produce small specific sequences of DNA. These are particularly important as primers in the polymerase chain reaction. 

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.

The polymerase chain reaction (PCR) is an important procedure in genetic engineering that allows any DNA segment to be replicated (amplified) without the need for restriction enzymes, vectors, or host cells (see p. 258). However, the nucleotide sequence of the segment has to be known. Two oligonucleotides (primers) are needed, which each hybridize with one of the strands at each end of the DNA segment to be amplified also needed are sufficient quantities of the four deoxyribonucleo-side triphosphates and a special heat-tolerant DNA polymerase. The primers are produced by chemical synthesis, and the polymerase is obtained from thermostable bacteria. 

It should be emphasized that the polymerase chain reaction requires specific primers for synthesis therefore, the sequence flanking the sequence to be amplified must be known. PCR reactions are also very susceptible to contamination by other DNA. Precautions against contamination need to be... 

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... 

The solid-phase synthesis of DNA oligomers has reached a high level of maturity and is extensively used for the synthesis of primers for polymerase chain reaction or the design of new genes (13). In contrast to the cellular process, the synthetic route forms the polymer in 3 -to-5 direction. The oligomerization is carried out on CPG using a base labile linker. Typically, the nucleobases are introduced as acyl-protected phosphoramidites,... 

Figure 6.8. The First Cycle in the Polymerase Chain Reaction (PCR). A cycle consists of three steps strand separation, hybridization of primers, and extension of primers by DNA synthesis.

Figure 5-47 Amplification of DNA using the polymerase chain reaction (PCR). Double-stranded DNA is denatured by heating to 90-99° C (step a) and oligonucleotide primers complementary to short 12-18 nucleotide sequences at the two ends of the piece of DNA to be amplified are annealed to the separated strands by cooling to 40 - 75° C (step b). The two DNA strands serve as templates for synthesis of new complementary strands using a heat-stable DNA polymerase and a mixture of the four nucleotide triphosphates. Nucleotide units are added to the 3 ends of the primers, with the new chains growing in the 5 3 ...

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