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DNA Consulting |
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DNA Profiling Methods: RFLP
Until a few years ago, most crime laboratories in the United States used a DNA profiling method known as Restriction Fragment Length Polymorphism (RFLP). Although RFLP yielded excellent results in many cases, the method was slow, labor intensive and required relatively large samples of high quality DNA. In the early days of RFLP, DNA profiles were detected with 32P labeled probes. It would often take 5 or 6 weeks to complete a single case. With the advent of enzyme linked probes and chemiluminescent detection in the mid 1990s, it was often possible to complete a simple case in 1 or 2 weeks.
RFLP Method:
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Isolate high molecular weight DNA from crime scene evidence and from reference samples |
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Cut DNA with a restriction enzyme (usually HaeIII) that does not cut within the VNTR elements |
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Apply DNA, along with size standards, to agarose gel |
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Electrophoresis: apply an electrical current across the length of the gel which will cause the negatively charged DNA fragments to separate according to their size |
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Transfer the separated DNA fragments to the surface of a nylon membrane |
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Hybridize membrane with an enzyme labeled DNA probe that will only bind to alleles for a specific locus. |
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Incubate the membrane with a chemiluminescent substrate. This substrate will emit light in the presence of the enzyme linked probe. |
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Expose to X-ray film |
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Remove probe and repeat with a probe for a different locus. |