Polymerase Chain Reaction [Published]

Mayberry

Polymerase Chain Reaction (PCR) is a type of DNA cloning where a specific DNA section is replicated multiple times to generate many identical copies of a certain sequence. It is one of the fastest ways to clone genes.

Before you can do anything, you must know about DNA sequences near the gene you are replicating. This will allow you to find appropriate primers to use in PCR. These primers must be complementary to bases on either side of the strand. In addition to this, you will also need an enzyme called Taq polymerase as it is able to function normally at the temperatures PCR is performed in. The last reactant you need is dNTP, with four varieties, each for a corresponding nucleobase (Adenosine, cytosine, thymine, and guanine.). This will provide the bases required to synthesize new DNA strands.


Summary of Materials:

- Template DNA strand
- Primers appropriate for the strand
- Taq polymerase (other polymerases are available)
- Four varieties of dNTP

Note: Other than the template DNA, have a generous amount of each material.


Instruments required:

- Reaction tubes (usually no more than 0.5 mL)
- Thermal cycler


Polymerase Chain Reaction:

1. Place all the materials into solution (~10-200 μL) in a reaction tube.
2. Place the reaction tube into the thermal cycler and heat to 94°C for several minutes. (This will denature the double-stranded DNA into single strands)
3. Cool the mixture to 50-60°C. (This is primer annealing. The primers will bind to the complementary sections of the DNA).
4. Heat the mixture to 72°C. (This is the ideal temp for the polymerase. It now synthesizes a complementary DNA strand).
5. One cycle of PCR has been complete. Repeat steps 2-4 as many times as needed.
6. Extract the DNA from the tubes for whatever purpose you require.

You have successfully completed a Polymerase Chain Reaction. For n number of cycles, you will have produced 2^n copies of the DNA sequence. In just 20 cycles, you will have over 1 million copies of the DNA!

This method is great for amplifying small amounts of DNA, like in bones of ancient animals. If enough DNA is intact, it can be replicated many times until enough is available for study.

Added to CMS by Trx100

Mayberry

I'll post some helpful pictures here as not to clutter the information.


Reaction tubes:




Thermal Cycler:

fanglekai

This all sounds really cool but I don't have any way of doing it or any use for the DNA. I want a jurassic park now, though.

Mayberry

fanglekai wrote: »

any use for the DNA. I want a jurassic park now, though.

There

busx

Had to write a paper on this in my freshman year, it's very simple and very clever! Excellent applications in crime labs. Ever wonder what they do with those tiny blood/skin samples?