A. Introduction to Polymerase Chain Reaction (PCR)

Mechanism of Recombination

In cloning a gene,  DNA must undergo a process to isolate the segment of interest so that it can be used as a template for cloning. Often times  when a gene is isolated, ligated into a plasmid DNA vector,  grown in bacterial colonies or libraries, and banked for research or sale it is inserted in a cloning vector. If the gene we intend to clone is contained in a cloning vector, a restriction enzyme reaction must be performed to remove the piece of DNA we intend to clone.   This segment is separated and purified from the rest of the recombinant plasmid components and amplified using the polymerase chain reaction.

 Restriction Reactions exploit the defense mechanism found in bacteria in its defense against viral infections.  Bacteria produce enzymes which have the ability to cut invading DNA, as the virus attempts to take over the host cell, to prevent infection.  They are named after the genus, species, and strain of bacteria they are found in. Restriction endonucleases  cut in predictable sites of nucleic acid sequences. As a result, this mechanism is used by scientist to target specific sequences of DNA they would like to isolate or remove from wild forms or engineered vectors. These reactions are performed in buffers that can hinder the amplification occurring in PCR so a purification process must take place to minimize competing reactions.  PCR  causes an exponential copying of an isolated and purified DNA segment or gene.  As a result we do not need a large amount of the gene isolated, since PCR assures we have abundant copies of the gene.

 Another use exploiting the predictable enzymatic cutting is in engineering restriction sites called poly-linkers within specific regions of bacterial plasmid DNA. This polylinker allows a scientist to use a variety of enzymes for blunt or forced cloning techniques. A PCR product containing an amplified gene or DNA segment is further purified, and undergoes a secondary restriction reaction using the same enzymes that cut an expression vector candidate chosen to receive the insert and form recombinant DNA. 

A Recombinant DNA plasmid is inserted into bacterial host cells using heat shock techniques. As cells divide, the mechanism of asexual reproduction allows replication of the inserted vector to pass the new recombinant DNA to emerging colonies. If the goal of cloning is just to insert and replicate recombinant DNA, a cloning vector is chosen as the candidate to receive the DNA insert. Vectors used in this way are called cloning vectors because they do not have the mechanism within them to express the protein of the inserted gene. If the expression of the protein encoded on the gene is desired an expression vector is selected as the plasmid candidate.  Such a plasmid can usually be induced to translate the gene using chemical applications, and expresses the recombinant protein. 

These genetic advances have been made possible through the development of the polymerase chain reaction.  PCR has facilitated cloning techniques, and created an avenue in which multidisciplinary sciences have joined together to tackle the genetic problems of our century. 
Subpages (2): History PCR Protocol