PCR and Electrophoresis

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PCR

Why replicate DNA sections?

Insulin

Favourable DNA

Forensics for analysis

PCR = Polymerase chain reaction

PCR

A technique that amplifies DNA allowing you to increase the amount of DNA from a small sample. It's important as it has many applications: forensics, genetic diseases, monitoring disease spread.

How does it work?

Artificial replication of DNA

Cyclic reaction

Needs:

DNA samples

DNA polymerase

Excess A, T, G, C

Primer - 3/4 base pairs long. Starts a bit of the double-strand, Allows DNA polymerase to work as doesn't work on single strands.

Thermal cycler

3 stages:

Denaturing - Heat DNA sample to 94-96 degrees to break hydrogen bonds. The whole strand unzips in denaturing.

Annealing - Cool to 68 degrees. This allows the primer to hydrogen bonds creating 1st new double-stranded DNA section.

Extending - Heat to 72 degrees, DNA polymerase binds to end and catalysts addition of nucleotides.

Uses of PCR:

Tissue typing

Detection of oncogenes (cancer genes)

Detecting mutations, e.g. Down syndrome

Identifying viral infections

Monitoring spread of infectious disease

Forensic science

Research

Detect ancient DNA

Cloning

Identifying transgenic plants

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Questions

1. Why are primers needed in PCR but not natural replication?

In PCR, DNA completely unzips. DNA polymerase only works on double strands, not single. A primer is 3/4 bases long and attaches to the single strand to start a double so polymerase can work. In natural it unzips slowly so there is always a double strand.

2. Why does DNA polymerase need to be extracted from T. aquaticus, from hot springs?

Otherwise DNA polymerase would denature at high temperatures due to the high temperatures.

T. aquaticus = Taq polymerase.

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Electrophoresis

Separates different size fragments of DNA using an electric current.

Uses:

DNA profiling

Paternity disputes

Analysing disease

Equipment

Electrophoresis tank

Agarose gel

Power supply

Buffer solution

Loading dye

Digested DNA

How does it work?

DNA cut into pieces

Gel placed in tank and the tank is filled with buffer.

Loading dye mixed with DNA, placed into well in jelly.

Electric current is passed through the gel

DNA carries a negative charge, as phosphate groups are negatives

Fragments migrate to anode (+)

Lighest fragments move fastest.

Do it for a set amount of time.

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Additional Information

What is the human genome?

It is all the genetic material an organism contains. Genes only make up 2% of human DNA, these are called exons. Introns are large non-coding regions of DNA which are removed from messenger RNA before it is translated into a polypeptide chain.