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The Future of Forensic DNA Testing

The Future of Forensic DNA Testing

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Published by: DOJ on Jan 22, 2008
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07/01/2013

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The Southern hybridization process is named after E.M. Southern, who developed and
published the method in 1975. Digestion of a human genomic DNA sample with a restric-
tion endonuclease generates a very large number of DNA fragments. Some are longer
than others. Subjecting the mixture to gel electrophoresis (A4.a, p. 50) generates a gra-
dation of fragments in the gel based on size, these classes being distinguished by their
length. Even so, each class contains a complex mixture of DNA fragments.

To determine the presence or absence of a particular fragment, such as a specific allele of
a VNTR locus, the separated DNA fragments from the sample are denatured, that is, sep-
arated into their two component strands, usually by treatment with alkali, and transferred
to a thinner, more sold support, a membrane, usually made of cellulose or nylon. The
membrane is exposed in a liquid solution to a probe, that is, a DNA sequence specific to
the VNTR locus. The probe is also single stranded, and mixing it under appropriate condi-
tions with the sample fragments allows hybridization between the sample DNA and probe
DNA, to occur only at the sites of complementary sequences (that is, where the sample
and probe sequences are the same except that where one has a T the other has an A,
and so on, so they match perfectly by Watson-Crick pairing rules). Thus, labels that are
attached to probes are also localized to the position(s) of the specific sample allele(s).

Probes generally contain either radioactive labels or an enzyme that can convert a sub-
strate into a luminescent output. In either case, the generated energy can be captured on
film to display the position of each allele. Sizes of the alleles can be compared with size
standards to assist with this process.

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