The Polymerase Chain Reaction (PCR)

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The Polymerase Chain Reaction (PCR)

It is artificial DNA replication.

Can be carried out on tiny samples of DNA in order to generate multiple copies of the sample. 

Particularly useful useful in forensic investigations, where DNA samples are taken from the crime scene, e.g. blood, can be multiplied in order to generate enough material for genetic profiling.

Sequencing relies one DNA being;

Made up of anti-parrallel backbone strands

Made up of strands that have a 5' prime and a 3' prime end.

Grows only from the 3' end

Base pairs pair up according to complementary base-pairing rules- A with T and C with G

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Not Identical to DNA replication

It can only replicate relatively short sequences of DNA (few hundred bases long), not entire chromosomes

The addition of primer molecules is requires in order for the precess to start

A cycle of heating and cooling is used in PCR to separate strands in the natural process

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It's a cyclic reaction

DNA sample is mixed with a supply of DNA nucleotides and DNA polymerase

Mixture is heated to 95°C which breaks hydrogen bonds holding the complementary bases, the sample becomes single stranded.

Primers or Short lengths of single-stranded DNA are added (10-20 bases) 

The temperature is reduced to 55°C, allowing primers to bind and form small double stranded sections of DNA at either end.

DNA Polymerase can bind to these double-stranded sections

DNA sample is mixed with a supply of DNA nucleotides and DNA polymerase

DNA polymerase is described as thermophilic, it isn't denatured at hight temperatures used in the process. It is derive from a thermophilic bacterium, Thermus aquaticus.

Mixture is heated to 95°C which breaks hydrogen bonds holding the complementary bases, the sample becomes single stranded.

Primers or Short lengths of single-stranded DNA are added (10-20 bases) 

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continued....

The temperature is reduced to 55°C, allowing primers to bind and form small double stranded sections of DNA at either end.

DNA Polymerase can bind to these double-stranded sections

The temperature is raised to 72°C (optimum temp for enzyme). The enzyme extends the double-stranded section by adding free nucleotides to the unwound DNA (same as natural DNA replication

When the DNA polymerase reaches the other end of the DNA strand, a new double-stranded DNA molecule is generated.

The whole process can be repeated many times so the amount of DNA increase exponentially

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Non-protein coding

They are known to code for the production of a variety of short mRNA strands.

Some are 'antisense' srands, bringing to the mRNA of other coding genes so that protein synthesus cannot take place.

Others bind to part of a coding mrNA and trigger destruction of the mRNA.

These processes are important in genome regulation.

The 'silencing' of genes in this way is the subject of much research-e.g. genes responsible  for laying down fat deposists have been artificially silenced in wormd by adding RNA that leads to destruction of the coding mRNA.

This artificial RNA interference is now known to have a natural role in genome control.

Cells can trim double-stranded RNA to form small inhibitory RNA or siRNA. An siRNA can be processed to the single-strand antisense RNA and used to target mRNAs for destuction. Several proteins are required for efficient RNA interference.

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