DNA Analysis: PCR and RFLP Techniques
By: Ingrid Hubata
Every organism has a set of DNA patterns that separate it from others, which form its genetic "fingerprint." DNA analysis techniques offer simple, quick screening. Currently, there are two primary types of forensic DNA testing, PCR and RFLP analysis.
GENERAL DNA OVERVIEW, INTRO TO PCR
Polymerase chain reaction (PCR) is a method used to increase the quantity of a particular section of DNA, so as to generate enough DNA to be tested. This method can be used to identify disease-causing viruses or bacteria, a deceased person, or a criminal suspect. The DNA of distinct organisms is different, and by identifying the genes that are different, one can use this information to identify an organism. The four components within a range of DNA are Adenine, Thymidine, Cytosine, and Guanine (A, T, C, and G). The arrangement of letters makes up a genetic sequence. DNA is double-stranded and the two strands pair up so that A always pairs with T, and C always pairs with G. Knowing the genetic sequence, one can successfully reproduce it. Only a very small amount of DNA is necessary to be able to use the PCR technique.
THE PCR TECHNIQUE
The PCR technique is performed by first heating the unknown DNA, (causing the paired strands to separate) then adding large excess of primers and cooling the reaction mixture to allow the strands to pair again. Then an enzyme (DNA polymerase) is added to the DNA that can read the opposing strands sequence and hook together (A-T, and C-G). Finally the enzyme synthesizes new DNA in opposite directions. This cycle gets repeated and produces plenty of DNA, which will be copies of the specific region. This technique of PCR allows accurate identification of the source of the copied DNA.
THE RFLP TECHNIQUE
Restriction Fragment Length Polymorphism (RFLP) DNA testing is a different type of analysis. The DNA is cut into fragments using a restriction enzyme that recognizes a particular genetic sequence that occurs repeatedly. The RFLP technique requires larger amounts of DNA, and the fragments are then separated by size on a gel through electrophoresis. The gel allows small DNA to move quicker than larger ones, with small DNA fragments near one end, and large ones near the other end. A copy of the gel's DNA is made on a blot, and the blot is then mixed with a piece of DNA that recognizes a particular genetic sequence. The blot is then placed on X-ray film. The radiation exposes the film, producing dark bands. The bands indicate the size of the fragments. The band sizes are measured by comparing them with known DNA sizes. If two samples tested are not the same length, then they must be from different people. However, if they are the same lengths then they either belong to the same person, or two different people who have fragments of the same length. These are descriptions of the two main types of forensic DNA testing. RFLP requires larger amounts of DNA than PCR, but crime evidence that is old is usually unsuitable for RFLP testing. PCR testing requires less DNA, and DNA that is partially degenerated is still suitable for PCR testing. However, both have sample size and degeneration conditions.
RELEVANT LINKS
http://falcon.cc.ukans.edu/~jbrown/pcr.htm
(What The Heck is PCR?)
http://www.scientific.org/tutorials/DNAEASY.html
(DNA Testing: An Introduction for Non-Scientists)