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DNA fingerprinting

Two different DNA tests exist, the RFLP and the PCR. Both are very accurate, but they're conducted in different ways.

RFLP - Restriction Fragment Length Polymorphism

  • more accurate than PCR
  • requires a large sample of fresh DNA
  • takes longer than PCR
  • when a match if found, there is no question as to whether the suspect was at the scene.

PCR - Polymerase Chain Reaction

  • not as accurate, takes less time, less costly
  • requires only a small sample
  • that sample is amplified using polymerases to force the small amounts of DNA to copy themselves many times
  • can be performed on older samples
  • PCR only analyzes a specific gene, which is analyzed to find matches
  • If no match is found, the donor was not at the scenes, however a match doesn't prove conclusively the person was there - many people may have the same match

Both PCR and RFLP require a second technique - known as Southern Blotting - which separates fragments of DNA and represents them as bands.

Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane.

Interestingly enough, the same procedure works on RNA, though instead of Southern Blotting, it is called Northern Blotting.

How Southern Blotting Works

 

Step 1: Isolation of DNA

DNA must be recovered from the cells of the plant or animal. Only a small amount is needed. For example, the amount of DNA found in one drop of blood or one square centimeter (about the size of a dime) of leaf tissue is usually sufficient. PCR or RFLP can be performed on this sample.

Step 2: Cutting, sizing, and sorting the DNA

Special enzymes called restriction enzymes are used to cut the DNA at specific places. For example, an enzyme called EcoRl found in bacteria will cut DNA only when the sequence GAATTC occurs.

The DNA pieces are sorted according to size by a electrophoresis through a gel made from seaweed agarose. Electrophoresis is the DNA equivalent of sieving sand through progressively finer mesh screens to determine particle size distribution.

Step 3: Transfer of DNA to nylon

The DNA pieces are transferred to a nylon sheet by placing the agarose gel and nylon next to each other overnight.

Steps 4-5: Probing

The DNA fingerprint is generated by adding tagged probes to the nylon sheet. Each probe typically sticks in only one or two specific places, wherever the sequences match (A with T and G with C). The tag allows detection of the probe.

Step 6: DNA fingerprint

The final DNA fingerprint is built from several different probes and resembles the bar codes used at the grocery counter.

Example of a Fingerprint used for Forensic Investigation

Terms

PCR
RFLP
Southern Blot
Gel electrophoresis
Restriction Enzyme
Probes
Agarose Gel
Nylon membrane

Links

Problem Sets in DNA Forensics
Problem Set 2