Nucleotide sequencing thermostable polymerase

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. 

An important technical difference between NGS techniques is whether the input DNA or RNA must be amplified in quantity before sequencing. This matters because the methods of amplifying DNA/ RNA can introduce errors in the sequence and also introduce biases in the sequences that are amplified. Typical thermostable polymerases, used for amplifying DNA/RNA in polymerase chain reactions (PCR), introduce mutations at a rate of 1 in 9000 nucleotides. This rate sounds rare, but the multiple amplification cycles necessarily (up to 40) can yield a number of sequence errors. Furthermore, particular types of sequences called microsatellites, such as long stretches of adenine nucleotides, may cause an even higher error rate. Likewise, polymerases often amplify GC-rich sequences at lower efficiency... 

PCR requires a thermostable DNA polymerase, nucleotides of each base (collectively referred to as dNTPs)y the target sequence, and a pair of oligonucleotides (referred to as primers) complementary to opposite strands flanking the sequence to be detected. In the first step, target duplexes are denatured into single strands by heat (Figure 37-2). When the mixture is cooled, primers provided in great excess (usually over a miUion times the concentration of the initial... 

The polymerase chain reaction (PCR) is a method to produce an exponentially growing number of copies from a DNA sequence which may very well be unknown, as long as short neighboring sequences are known and can be synthesized synthetically to be used as primers (Fig. 2.10). Of each strand, a short sequence upstream of the sequence of interest (i.e. located in 5 direction) must be synthesized. These primers are added to the DNA, which is mixed with a thermostable DNA-polymerase (obtained from heat-loving bacteria, or nowadays from any provider of biochemical products) and the nucleotide triphosphates, the raw materials in the synthesis of DNA. 

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