PCR, Restriction Enzymes and Genomic Libraries

Hi :) Today's post will cover the next topic in our Veterinary Biochemistry unit - molecular tools. In this post I'll be discussing the Polymerase Chain Reaction (PCR) technique, restriction enzyme and how genomic libraries are constructed. 

Polymerase Chain Reaction (PCR):

The PCR technique is the amplification of nucleic acid sequences via repeated cycles of:

1. denaturation,
2. oligonucleotide primer annealing,
3. and DNA polymerase extension using a heat stable polymerase. 

PCR generates billions of copies of target DNA or RNA. 
The components of a PCR reaction include: the DNA template; primers; dNTPs (nucleotides) including dATP, dTTP, dCTP, dGTP; and DNA polymerase (a heat stable DNA polymerase taken from thermophilic bacteria). 


The process is summarised really well in the video below:

Advantages of PCR:

The PCR reaction is a very specific assay, this is determined by the two primers used. PCR also allows you to start with very small amounts of DNA. This means that it can be used to analyse single cells or to amplify the starting material. PCR is not dependent on the DNA being intact, this is useful for forensic analysis. PCR only generates products when the binding sites for primers are present. This allows one to directly detect the presence of mutations because mutations prevent binding. This also allows PCR to be used as a diagnostic technique for the presence of foreign DNA. PCR can also be used in combination with other methods to amplify the amount of DNA available for analysis. 

Restriction Enzymes

Restriction enzymes are like DNA scissors. They recognise short DNA sequences and cut the DNA molecules at those specific sites. Their natural biological function is to protect bacteria by attacking viral and other foreign DNA. Some restriction enzymes make staggered cut that leave the resulting DNA with sticky ends. Enzymes which cleave the DNA at the centre of the recognition sequence leave blunt ended fragments of DNA. 

Genomic Libraries 


Genomic DNA libraries contain all the DNA of an organism. They are usually constructed for whole genome sequencing projects. There are 4 steps involved in constructing a DNA library:

1. Extract and purify genomic DNA
2. Digest DNA with restriction enzyme
3. Insert into a suitable vector (eg. plasmid) and seal DNA with suitable ligase
4. Plasmids containing foreign DNA are introduced (transformed) into bacteria and allowed to grow.

The plasmids which are in the bacteria give the bacteria resistance to antibiotics. This allows the particular bacteria to be isolated from other bacteria in a sample.