Saturday, June 22, 2013

DNA Fingerprinting

DNA Fingerprinting also called DNA typing or DNA profiling:

DNA fingerprinting is based on sequence polymorphisms, slight sequence differences between individuals, 1 bp in every 1,000 bp, on average. Every individual has some differences. Some of the sequence change affects 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 most commonly in DNA typing involves short tandem repeats (STRs). The detection of RFLPs relies on Southern blotting.

In the final x-ray film suspect 1 and evidence banding pattern are similar.

Although accurate, due to need of large sample of undegraded DNA (>25 ng). that amount is often not available at a crime scene or disaster site.

The search for more sensitive method led to develop 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 tetranucleotide repeats), and have multiple length variants in human population. More than 20, 000 tetranucleotide STR loci have been found in human genome. More than a million STRs of all types may be present in a human genome, accounting for about 3% of all human DNA.

The polymerase chain reaction is applied to STR analysis, and focused mainly on STRs as they are very  sensitive.  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.

PCR primers can amplify a segment of DNA containing repeat region. One of the two primers is linked to fluorescent dye (green circle). Since the two chromosomes has different allele, thus have different number of repeats at same locus. The PCR thus generate two products of slightly different size. These are subjected to electrophoresis on thin PAGE in capillary tube. The resulting fluorescent bands are scanned by laser to generate series of peaks, that when combined with appropriate size marker controls, accurately reveals the size of each PCR fragment and thus the length of STR in corresponding allele.

The U.S. standard, called the COmbined DNA lndex System (CODIS), established in 1998 is based on 13 well-studied STR loci, 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.

Convenient kits have been developed commercially that allow the amplification of 16 or more STR loci in one test tube. These "multiplex" STR kits have PCR primers unique to each locus. Each primer is carefully designed to avoid hybridization to any other primer in the kit and to generate PCR products of different sizes that spread out as signals from different loci during electrophoresis. The primers are Iinked to colored dyes to help distinguish the different PCR products. When good DNA profiles are obtained, the chance of an accidental match between two individuals in the human population is less than 1 in 1018 (quintillion). DNA typing has been used to both convict and acquit suspects, establish paternity with certainty. Even very old mysteries can be solved: in 1996, DNA fingerprinting helped to confirm the identification of the bones of the last Russian czar and his family, who were assassinated in 1918.

Combination of PCR products from multiple loci generates a multiplex pattern such as shown (here a pattern with 16 loci, called a 16-plex generated using a commercial STR analysis kit). This requires multiple sets of PCR primers, with one set targeted to each locus.

(Source: Lehninger's Textbook of Biochemistry and Lippincott's Illustrated Biochemistry)

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