Genetic fingerprinting catches on

Genetic fingerprinting catches on A CENTRE for DNA Fingerprinting and Diagnostics has been mooted by scientists at the Hyderabad-based Centre for Cellular and Molecular Biology (CCMB) to help forensic experts establish the identity of criminals beyond reasonable doubt. But the proposal is caught in a tangle between the Department of Biotechnology (DBT), the home ministry and the Council of Scientific and Industrial Research in Delhi.

The DBT is keen to keep the Centre under its control, but molecular biologist Lalji Singh of CCMB, who pioneered the DNA fingerprinting technique in India, insists it should be an autonomous body to prevent undue pressure on the scientists. Though Rs 50 lakh has been released towards planning the Centre, the legal documents have yet to be signed.

Singh is among the few scientists in the world who have developed probes or markers to locate genetic patterns (fingerprints) which are unique to each individual.

Singh has helped forensic experts identify criminals and establish paternity in more than 60 legal cases. The identities of former prime minister Rajiv Gandhi's assassin, Dhanu, and her accomplice, Sivarasan, were confirmed through DNA fingerprinting. It established that Dhanu wore the belt-bomb that killed her and Gandhi. The DNA fingerprints obtained from the remnants of her body found flung all over the site of the explosion were compared with the tissues attached to the belt to confirm this.

Says Singh, "This is an extremely powerful technique. Using DNA fingerprinting, one can be 99.99 per cent sure of a person's identity." But with the popularity of this method increasing, Singh says, "it is becoming humanly impossible for the small staff at CCMB to handle all the requests that come in from across the country. The proposed centre will strengthen the hands of the law to dispense justice and it is imperative that it comes up soon."

Singh recalls, "In the first case, in 1988, before the chief judicial magistrate of Tellicherry, I presented evidence to a packed court for some two to three hours." In this paternity dispute, Kunhiraman, a wealthy bachelor was accused by E Vilasini of being the father of her son, Manoj. But Kunhiraman denied paternity. Vilasini approached CCMB which found that every band in Manoj's DNA profile was derived either from Kunhiraman or Vilasini. The chance that Kunhiraman and Manoj shared the same bands but were unrelated was one in 300 billion. The court ruled that they were in fact father and son.

In another case, Singh was able to confirm that a few bones found in a eucalyptus grove in Bangalore belonged to Beena, a young woman who was murdered and chopped to bits by her husband, M V Mahesh, and his father, M Vishakantaiah, a university professor. Beena, suspected of adultery, was given cyanide. Star witness, servant Subba, led the police to Beena's remains, but turned hostile in court. To prove that the bones belonged to Beena, Singh extracted DNA from the bones and compared it with blood samples of Beena's mother, sister and brother. The similarity was striking.

Genetic fingerprinting is based on repetitive patterns in DNA -- the molecular structure which is unique to each individual and which carries genetic information from one generation to the next. DNA is a complex chemical with a double standard helical structure, which is made up of a number of bases namely, adenine (A), thymine (T), guanine (G) and cytosine (C), attached to sugar and phosphates. The bases present on each strand link with specific bases on the opposite strand of the helix. Guanine always links with cytosine and adenine with thymine. These arrangements comprise the genetic code and are unique to an individual. Genes are nothing but different combinations of these bases. Encoded in the human DNA are some 10,000 genes which represent only 5 per cent of the total DNA, made up of some three billion base pairs. The function of the remaining 95 per cent of the human DNA is not understood and is variously called "junk" DNA, "nonsense" DNA or "ignorant" DNA. This "junk" DNA consists of sets of base sequences repeated several times called minisatellites.

"This," says Singh, "may be responsible for the variation amongst human beings." Scientists have found that a great deal of variation occurs in the number of repeated units and thus, the length of the minisatellite, which is unique to each individual. With the recent development of genetic probes, scientists can study this variation at a molecular level.

The first genetic fingerprinting probe was developed by Alex Jeffrey at Leicester University in UK in 1985 and subsequently, Singh developed another one with the help of DNA from the sex chromosome of the female Indian banded krait. Using this probe called Bkm, Singh and his colleagues have recovered clones from the human and mouse genomes. Probes make use of the basic link characteristics of DNA -- that is, G will only link with C. The radioactively labelled probe of known bases seeks out matching sequences in a thicket of DNA and hybridises with them. This is then detected by autoradiography on an x-ray plate.

To undertake genetic fingerprinting, DNA of high molecular weight is isolated from blood or semen stains and vaginal swabs. DNA is then cleaved with "chemical scissors" -- enzymes which cut DNA at specific places -- and the fragments generated are separated according to their molecular weight. The DNA fragments are then transferred to a nylon membrane and hybridised with the radioactive Bkm probe in solution at 600C. The autoradiograph, which indicates areas of hybridisation -- where the probe links with the DNA -- is the DNA fingerprint.

Identical twins have the same DNA fingerprints, but fingerprints of fraternal twins are very different. The identity of criminals can be established if a complete DNA fingerprint match is obtained between the suspect's blood and a sample found at the crime site.

With the growing interest in wild crop germplasm, this technique can also be used to identify the seed stock from which the economically useful genes were obtained.

Imported probes are expensive, says Singh. Jeffrey's probe is being manufactured by ICI Diagnostics, a British firm, which has set up a centre called Cellmark Diagnostic to provide DNA fingerprinting services. It charges Rs 42,000 for just isolating and profiling human DNA.

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