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How was the Human Genome established? How was our DNA sequence determined? The details of DNA sequencing methods are presented in this section. They are Polymerase Chain Reaction, Restriction Fragment Length Polymorphism and Southern Blotting.

PCR

RFLP

Southern Blotting

PCR (Polymerase Chain Reaction)

Polymerase chain reaction is one of the most powerful molecular biology techniques developed to date for DNA amplification and investigation. The procedure amplifies minute or trace amounts of DNA by multiple cycles of cooling and heating in a reaction catalyzed by a heat stable DNA polymerase enzyme. Numerous copies of the target DNA can be made in a relatively short time. The DNA produced can be sequenced, analyzed, mapped with endonucleases, separated by electrophoresis, cloned,and characterized.


Old Faithful Geyser - Yellowstone National Park

An alien and inhospitable place, characterized by extreme heat, spewing steam into the frigid air such is the scene of old faithful geyser in Yellowstone National park. This seemingly prohibitive habitat is home to Thermus aquaticus, the bacterium from which the heat stable DNA polymerase essential to PCR is obtained.


Kary Mullis accepting the Nobel Prize

Brilliant, gifted, genius, rebel Dr Kary Mullis has been thus described, and more. Mullis discovered the PCR procedure, for which he was awarded the Nobel prize in 1993. The procedure he devised has revolutionized molecular biology, forensics, and DNA based identification technology.

PCR is a DNA amplification procedure in which numerous copies of DNA are generated from initial minute/trace amounts. The DNA is first heated to separate the strands of the double helix. Nucleotides(shown here in blue, orange) are added to the PCR reaction mix, along with short pieces of DNA called primers. These primers match the original DNA sequence being amplified. Heat stable DNA polymerase(Taq.DNA polymerase ) is added, and the mix is subjected to several cycles of replication in which new DNA strands are made by adding nucleotides to the primers. DNA helices made in each cycle are separated by heating to serve as templates for making more new DNA. Billions of copies of the original DNA can be generated in a few hours after several cycles of replication in a computer controlled thermocycler providing alternate cycles of heating and cooling.


Move your mouse over the image above to see the animation.

Copyright © M.A. Gilles-Gonzalez, Ph.D.


RFLP (Restriction Fragment Length Polymorphism)

Each individual has a unique and characteristic combination of nucleotides in their DNA. This unique combination can be analyzed and used for definitive identification. To do this, the person’s DNA is first cut into small fragments by the action of restriction endonucleases. These are enzymes (derived from bacteria largely) which cut DNA into pieces based on unique sequences recognized by the different enzymes. Different pieces of DNA are produced by using combinations of different enzymes, and the pieces produced by each person’s DNA are unique. The DNA fragments produced are then separated by gel electrophoresis to produce patterns characteristic of the person from which the DNA was obtained.

 

 


Southern Blotting

Southern blotting may be defined as the elution of DNA fragments (from gels after electrophoresis) to nitrocellulose or nylon membranes. The DNA blotted or transferred to the membrane can then be detected by hybridization (binding) with a labeled probe. Binding of the probe is detected using radioactive label, chemiluminescense, or colorimetric methods. DNA fragments are produced by restriction endonuclease digest of target DNA

This procedure allows detection of various DNA gene sequences, and is one of the most widely used procedures in molecular biology.

 


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