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1. Beskrive Polymerase Chain Reaction (PCR), og hvordan den kan bruges til at amplificere et stykke DNA
 Devlin, s. 281, fig. 7.1
Stryer, s.149-150, fig. 6.8, fig 6.9

 

Invented in 1984 by Mullis.

PCR is used for amplifying specific DNA sequences.

Consider a DNA duplex consisting of target sequence surrounded by non-target DNA. Millions of the target sequences can be readily obtained by PCR if the flanking sequences of the target are known.  

The following components are required for PCR:

  1. a pair of primers that hybridise with the flanking sequence of the target

  2. all four deoxyribonuclease triphosphates

  3. a heat stable DNA polymerase

 

A PCR cycle consists of three steps:

  1. Strand separation – the two strands of the parent DNA molecule are separated by heating the solution to 95˚C for 15 s
     

  2. Hybridization of primers – the solution is then abruptly cooled to 54˚C to allow the primers to hybridize to the flanking (non-target) region of the DNA strand. One primer hybridizes to the 3’-end of the target on one strand, and other primer hybridizes to the 3’-end on the complementary target strand. Parent DNA duplexes do not form, because the primers are present in large excess. Primers are typically from 20-30 nucleotides long.   
     

  3. DNA synthesis – the solution is then heated to 72˚C, the optimal temperature for Taq DNA-polymerase. This heat stable DNA polymerase comes from Thermus Aquaticus, a thermophillic bacterium that lives in hot springs. The polymerase elongates both primers in the 5’-3’ direction, the direction of the target sequence. DNA synthesis takes place on both strands, but extends beyond the target sequence.

 

This is the first cycle of PCR, creating two new target DNA-copies, but they also extend beyond the target sequence. We only need the target sequence.

The second cycle has the same steps as the first one, and at the end, we get four new target DNA-sequences, two extending beyond the target sequence and two that are not, due to the fact that the primer had hybridized with one of the two newly synthesised DNA-target sequences from the first cycle, and they do not extend beyond the 3’-end where the original primer was bounded. These target DNA sequences are called short strands.

At the end of the third cycle, these two short strands have replicated, so we get two double stranded target molecules. The third cycle is therefore the first cycle that produces double stranded target molecules. They are used for further target DNA sequence synthesis.

The large strands, the ones that extend beyond the target sequence are still in the solution and are used for providing more short strands.

Ideally, after n cycles, the sequence is amplified 2n – fold.

So after 22 cycles there over 1.000.000 double stranded target molecules in the solution; after 32 cycles - over 1.000.000.000.

Few noteworthy facts about PCR:

  1. the sequence of the target DNA need not be known, all that is necessary is the knowledge of the flanking regions

  2. the target can be much longer then the primer

  3. primers need not be perfectly matched to the flanking sequences

A target DNA can be inserted into a vector of known sequence by recombination technologies.

 

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