June 17, 2011

Polymerase Chain Reaction (PCR)



Introduction:

            The molecular technique called PCR is in vitro amplification of a specific segment of DNA using a thermostable enzyme.  Although it is a fairly new technique, invented in 1985 by Cary Mullis, it is widely used in hundreds of labs all over the world.  The PCR process makes millions of copies of DNA in just a few hours.  It is a replication reaction, which uses reagents very similar to what is needed for DNA replication inside a cell.  Each strand serves as a template for synthesis of its complementary strand.

            PCR has many applications. 1) It is a form of direct cloning of DNA (without the need for bacteria).  This is convenient when there is little DNA to work with.  2) PCR can produce a DNA fingerprinting pattern for forensics purposes, such as identifying blood at a crime scene.  3) PCR can be used in prenatal diagnosis of genetic diseases. 4) It can be used for evolutionary analyses, to look at genetic relationships among or within taxa (genera, species, populations).  5) PCR can detect allelic sequence variation and chromosomal rearrangements.  6) It is involved in the DNA sequencing process.  7) It is the newest technique in detecting viral or bacterial infection within a host. 

Gel Electrophoresis



Introduction:

The size of a DNA fragment can be estimated by gel electrophoresis.  This technique separates fragments by charge, size (molecular weight) and shape.  First, an agarose gel is made with slots (wells) in it.  The DNA sample is dispensed in the well, a buffer solution is placed in the apparatus, and an electric current is run through the gel.   DNA molecules are negatively charged due to the phosphates in its backbone, and when placed in an electric field starting at the negative (black) electrode, it will migrate towards the positive (red) electrode.  The gel is a complex molecular network containing narrow passages.  Smaller DNA molecules pass through more easily (less friction) and migrate faster through the gel than larger size fragments.   Linear molecules also migrate faster through a gel, compared to globular  forms.  

            Since the DNA fragments generated in this experiment are all negatively charged and linear in shape, the only variable to observe during electrophoresis is size.  To determine the size of a molecule, a standard or positive control is run concurrently in the gel.  The standard consists of fragments of DNA of known size.  A positive control contains the same size fragment (in our case 662 bp) as potential positive samples.