DNA FINGERPRINTING

DNA fingerprinting/ DNA typing/ DNA profiling/ DNA test

It is technique to identify a person on the basis of his/her DNA specificity.

This technique was invented by sir Alec. Jeffery (1984).

In India DNA Finger printing has been started by Dr. V.K. Kashyap & Dr. Lal Ji Singh.

DNA of human is almost the same for all individuals but very small amount that differs from person to person that forensic scientists analyze to identify people.

These differences are called Polymorphism (many forms) and are the key of DNA typing. Polymorphisms are

most useful to forensic scientist. It is consist of variation in the length of DNA at specific loci is called Restricted fragment. It is most important segment for DNA test made up of short repetitive nucleotide sequences, these are called VNTRs (variable number of tandem repeat).

VNTRs also called minisatellites were discovered by Alec Jeffery. Restricted fragment consist of hypervariable repeat region of DNA having a basic repeat sequence of 11-60 bp and flanked on both sites by restriction site.

The number and position of minisatellites or VNTR in restriction fragment is different for each DNA and length of restriction fragment is depend on number of VNTR.

Therefore, when the genome of two people are cut using the same restriction enzyme the length of fragments obtained is different for both the people.

These variations in length of restricted fragment is called RFLP or Restriction fragment length polymorphism.

Restriction Fragment Length Polymorphism distributed throughout human genomes are useful for DNA Fingerprinting.

 DNA Fingerprint can be prepared from extremely minute amount of blood, semen, hair bulb or any other cell of the body.

DNA content of 1 - Microgram is sufficient.

Technique of DNA Fingerprinting involves the following major steps.

Extraction - DNA extracted from the cell by cell lysis. If the content of DNA is limited then DNA can be amplified by Polymerase chain reaction (PCR). This process is amplification.

Restriction Enzyme Digestion : Restriction enzyme cuts DNA at specific 4 or 6 base pair sequences called restriction site.

 Hae III (Haemophilus aegyptius) is most commonly used enzyme. It cuts the DNA, every where the bases are arranged in the sequence GGCC. These restriction fragment transferred to Agarose Polymer gel.

Gel Electrophoresis :

Gel electrophoresis is a method that separates macromolecules either nucleic acid or proteins-on the basis of size, electric charge.

Gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provides the driving force. A molecule's properties determine, how rapidly an electric field can move the molecule through a gelatinous medium.

Nowadays the most commonly used matrix is agarose which is a natural polymer extracted from sea weeds. The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel.

Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids posses ionisable groups and therefore, at any given pH, exist in solution as electrically charged species either as cation (+) or anions (-). Depending on the nature of the net charge, the charged particles will migrate either to the cathode or to the anode.

By the gel electrophoresis these restricted fragments move towards the positive electrode (anode) because DNA  has -ve electric charge (PO₄³⁻).

Smaller Fragment more move towards the positive pole due to less molecular weight. So after the gel electrophoresis DNA fragment arranged according to molecular weight.

These separated fragments can be visualized by staining them with a dye that fluoresces ultraviolet radiation.

Southern transfer / Southern blotting :

The gel is fragile. It is necessary to remove the DNA from the gel and permanently attaches it to a solid support. This is accomplished by the process of Southern blotting. The first step is to denature the DNA in the gel which means that the double-stranded restriction fragments are chemically separated into the single stranded form.

The DNA then is transferred by the process of blotting to a sheet of nylon. The nylon acts like an ink blotter and "blots" up the separated DNA fragments, the restriction fragments, invisible at this stage are irreversibly attached to the nylon membrane the "blot".

This process is called Southern blot by the name of Edward Southern (1970).

Hybridization: To detect VNTR locus on restricted fragment, we use single stranded Radioactive Phosphorus-32 (P32) DNA probe which have the base pair sequences complimentary to the DNA sequences at the VNTR locus. Commonly we use a combination of at least 4 to 6 separate DNA probes.

Labelled Probes are attached with the VNTR loci of restricted DNA Fragments, this process is called Hybridization.

Autoradiography: Nylon membrane containing radio active probe exposed to X-ray. Specific bands appear on X-ray film. These bands are the areas where the radioactive probe bind with the VNTR.

This appears the specific restriction fragment length pattern. This length pattern is different in different individual. This is called Restriction Fragment length Polymorphism (RFLP).

These allow analyzer to identify a particular person DNA, the occurrence and frequency of a particular genetic patter contained in this X-ray film. These x-ray film called DNA signature of a person which is specific for each individual.

The probability of two unrelated individual having same pattern of location and repeat number of minisatellite (VNTR) is one in ten billion (world population 6.1 billion).

In India the centre for DNA fingerprinting and diagnosis (CDFD - center for DNA finger printing & diagnosis) located at Hyderabad.

Application of DNA Fingerprinting

1. Paternity tests- The major application of DNA finger printing is in determining family relationships. For identifying the true (biological) father, DNA samples of Child, mother and possible fathers are taken and their DNA finger prints are obtained. The prints of child DNA match to the prints of biological parents.

2. Identification of the criminal- DNA fingerprinting has now become useful technique in forensic (crime detecting) science, specially when serious crimes such as murders and rapes are involved. For identifying a criminal, the DNA fingerprints of the suspects from blood or hair or semen picked up from the scene of crime are prepared and compared. The DNA fingerprint of the person matching the one obtained from sample collected from scene of crime can give a clue to the actual criminal.

schematic representation of DNA fingerprinting: few representative chromosomes have been shown to contain different copy number of VNTR. For the sake of understanding colour schemes have been used to trace the origin of each band in the gel. The two alleles (parental and maternal) of chromosome also contain different copy numbers of VNTR. It is clear that the banding pattern of DNA from crime sceme matches with individual B, and not with A.

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