PCR and protein expression

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. 

the starter is heated to around 90 °C to separate the DNA double helix into single strands (a cfp. 84). The mixture is then cooled to allow hybridization of the primers (b). Starting from the primers, complementary DNA strands are now synthesized in both directions by the polymerase (c). This cycle (cycle 1) is repeated 20-30 times with the same reaction mixture (cycle 2 and subsequent cycles). The cyclic heating and cooling are carried out by computer-controlled thermostats. 

After only the third cycle, double strands start to form with a length equal to the distance between the two primers. The proportion of these approximately doubles during each cycle, until almost all of the newly synthesized segments have the correct length. 

Koolman, Color Atlas of Biochemistry, 2nd edition 2005 Thieme All rights reserved. Usage subject to terms and conditions of license. 

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