Traditionally, one of the most accurate methods for placing an individual at the scene of a crime has been a fingerprint. With the advent of recombinant DNA technology, a more powerful tool is now available: DNA fingerprinting (also called DNA typing or DNA profiling). The method was first described by English geneticist Alec Jeffreys in 1985.
DNA fingerprinting is based on sequence polymorphisms, slight sequence differences between individuals, 1 bp in every 1,000 bp, on average. Each difference from the prototype human genome sequence (the first one obtained) occurs in some fraction of the human population; every individual has some differences. Some of the sequence changes affect recognition sites for restriction enzymes, resulting in variation in the size of DNA fragments produced by digestion with a particular restriction enzyme. These variations are restriction fragment length polymorphisms (RFLPs). Another type of sequence variation, and the one now used most commonly in DNA typing, involves short tandem repeats (STRs).
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The detection of RFLPs relies on a specialized hybridization procedure called Southern blotting (Fig). DNA fragments from digestion of genomic DNA by restriction endonucleases are separated by size electrophoretically, denatured by soaking the agarose gel in alkali, and then blotted onto a nylon membrane to reproduce the distribution of fragments in the gel. The membrane is immersed in a solution containing a radioactively labeled DNA probe. A probe for a sequence that is repeated several times in the human genome generally identifies a few of the thousands of DNA fragments generated when the human genome is digested with a restriction endonuclease. Auto-radiography reveals the fragments to which the probe hybridizes, as in Figure. The method is very accurate and was first used in court cases in the late 1980s. However, it requires a large sample of undegraded DNA(25 ng). That amount of DNA is often not available at a crime scene or disaster site.
The requirement for more-sensitive DNA typing methods led to a focus on the polymerase chain reaction (PCR), and on STRs. An STR locus is a short DNA sequence, repeated many times in tandem at a particular location in a chromosome; most commonly, the repeated sequences are 4 bp long. The STR loci that are most useful for DNA typing are quite short, from 4 to 50 repeats long (16 to 200 total base pairs for tetra-nucleotide repeats), and have multiple length variants in the human population. More than 20,000 tetra-nucleotide STR loci have been characterized in the human genome. More than a million STRs of all types may be present in the human genome, accounting for about 3% of all human DNA.
The polymerase chain reaction is readily applied to STR analysis, and the focus of forensic scientists changed from RFLPs to STRs as the promise of increased sensitivity became apparent in the early 1990s. The DNA sequences flanking STRs are unique to each type of STR and identical (except for very rare mutations) in all humans. PCR primers are targeted to this flanking DNA, and designed to amplify the DNA across the STR. The length of the PCR product then reflects the length of the STR in that sample. Since each human inherits one chromosome from each parent, the STR lengths on the two chromosomes are often different, generating two signals from one individual. If multiple STR loci are analyzed, a profile can be generated that is essentially unique to a particular individual. PCR amplification allows investigators to obtain DNA fingerprints from less than 1 ng of partially degraded DNA, an amount that can be obtained from a single hair follicle, a drop of blood, a small semen sample on a bed sheet, or samples that might be months or even many years old.
Successful forensic use of STR analysis required standardization. The first forensic STR standard was established in the United Kingdom in 1995. The U.S. standard, called the COmbined DNA Index System (CODIS), was established in 1998. The CODIS system is based on 13 well-studied STR loci (Table), which must be present in any DNA typing experiment carried out in the United States. The amelogenin gene is also used as a marker. This gene, present on the human sex chromosomes, has slightly different flanking DNA on the X and Y chromosomes. PCR amplification across the amelogenin gene thus generates different-size products that can reveal the sex of the DNA donor.
The polymerase chain reaction is readily applied to STR analysis, and the focus of forensic scientists changed from RFLPs to STRs as the promise of increased sensitivity became apparent in the early 1990s. The DNA sequences flanking STRs are unique to each type of STR and identical (except for very rare mutations) in all humans. PCR primers are targeted to this flanking DNA, and designed to amplify the DNA across the STR. The length of the PCR product then reflects the length of the STR in that sample. Since each human inherits one chromosome from each parent, the STR lengths on the two chromosomes are often different, generating two signals from one individual. If multiple STR loci are analyzed, a profile can be generated that is essentially unique to a particular individual. PCR amplification allows investigators to obtain DNA fingerprints from less than 1 ng of partially degraded DNA, an amount that can be obtained from a single hair follicle, a drop of blood, a small semen sample on a bed sheet, or samples that might be months or even many years old.
Successful forensic use of STR analysis required standardization. The first forensic STR standard was established in the United Kingdom in 1995. The U.S. standard, called the COmbined DNA Index System (CODIS), was established in 1998. The CODIS system is based on 13 well-studied STR loci (Table), which must be present in any DNA typing experiment carried out in the United States. The amelogenin gene is also used as a marker. This gene, present on the human sex chromosomes, has slightly different flanking DNA on the X and Y chromosomes. PCR amplification across the amelogenin gene thus generates different-size products that can reveal the sex of the DNA donor.
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Authored and Published by;
Raj Abhisek Panda
Source:(1) Adapted from Butler, J.M. (2005) Forensic DNA Typing, 2nd edn, Academic Press, San Diego, p. 96.
(2) LEHNINGER PRINCIPLES OF BIOCHEMISTRY(FIFTH EDITION),Author-David L. Nelson, Michael M. Cox, Production-W. H. Freeman and Company, 41 Madison Avenue, New York, NY 10010
*Repeat lengths observed in the human population. Partial or imperfect repeats can be included in some alleles.
*Repeat lengths observed in the human population. Partial or imperfect repeats can be included in some alleles.
*Number of different alleles observed to date in the human population. Careful analysis of a locus in many individuals is a prerequisite to its use in forensic DNA typing.
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