Method 1 RFLP Analysis

The most common method of DNA typing is RFLP analyisis of VNTR loci.

 

 

Following standard protocols DNA is extracted and purified. It is cut into fragments using enzymes called restriction endonucleases, which recognize short DNA sequences and cleave only at or near specific tandemly repeated base sequences. Such areas, known as restriction enzyme recognition sites, occur on either side of the polymorphic locus of interest. Since the DNA fragments are of variable lengths, they are referred to as restriction fragment length polymorphisms (RFLPs). These fragments are sorted on the basis of size by electrophoresis in an agarose gel. This involves applying an electric current through a gel with the current traveling from the negative electrode at the top of the gel to the positive electrode at the bottom of the gel. The DNA fragments are negatively charged due to the phosphate groups and therefore travel toward the positive electrode. The smallest fragments migrate to the bottom and the largest remain on top. DNA is made single-stranded or denatured and a nylon membrane is placed on top of the gel. DNA is then transferred to the membrane via capillary action and fixed by baking making it accessible to a probe. A radioactive probe specifically binds to appropriate VNTR fragments by complementary base pairing. X-ray film is placed over the membrane and the DNA profile visualized by auto-radiography.

 

SUMMARY

Step A - Separation of nucleic acids band on size by agarose gel electrophoresis.

Step B & C - Transfer of separated nucleic acids from gel to nitrocellulose paper.

Step D - Hybridization with radioactive probe.

Step E - Auto-radiograph of DNA profile.

 

This process, known as the Southern Blot Technique, was developed in 1975 and adopted by Jeffreys et al for RFLP analysis for the first DNA fingerprinting in 1985. The DNA banding patterns were difficult to interpret as he used probes containing core sequences of base pairs that identified tandem repeats on many different chromosomes. The following year the single locus probe method was introduced becoming the preferred method.

 

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