Tracing truth
IT is not so long since scientists claimed to have deciphered the code of life: the deoxyribonucleic acid. DNA, as it is commonly known, had foxed scientists ever since it was discovered as the key player in genetic transfer of characters in all forms of life.
Interestingly, close on the heels of the breakthrough early this year, newspaper readers in India were deluged with reports of IDNA fingerprinting' as a method of identifying a virtually unidentifiable body of a murdered politician, Naina Sahni.
The body had been found in an advanced state of decomposition inside a tandoor (earthen oven), mutilated and burnt beyond recognition. The police needed a confirmation of identity from the woman's parents, who refused to oblige. The cops then resorted to DNA fingerprinting.
Scientific developments have always aided crime fighting (see box: Family tree). DNA profiling is the latest of such examples. Nature, in all its uniqueness, gives every individual of a species its own signature, the mark of its excluisivity. In simple terms, 'DNA fingerprinting' (which is really the lay usage for the scientifically correct term, 'DNA profiling'), is used as a method of establishing the identity of an unidentified body by tracing and matching these 'signatures' peculiar to an individual of a species.
A highly stable bio-chernical molecule, DNA is found in the nucleus of the white blood cells only, present in the tissues, bone marrow, roots of the hair, and in the case of males, in the semen. The acid does not loose its characteristics over millennia. Climate and the immediate atmosphere can lead to bacterial contamination of a cell, causing a certain extent of degradation. The chances of positive identification, however, are not entirely lost. This was proved When the method was used to draw DNA profiles of mummies recovered from Egyptian tombs, or even those of the dinosaurs.
"While the period from the time of a person's demise is not the most important factor, the quantity of samples collected is of vital importance," states N Guna Chandran, of the National Institute of Criminology and Forensic Science, Delhi, adding that if the recovered samples of blood, hair, bone or tissue were exceedingly small, analysis becames difficult.
However, there is a process by which even minute quantities Of DNA can be amplified, using the Polymerase Chain Reaction (PCR) method. The PCR is a thermal cycler, alternating between heating and cooling DNA fragments.
The sample fragments recovered are mixed with reagents and heated. The DNA strands get separa-ted, and the complimantary pairs come together during the cooling process. The 'harvest' is thus improved, with thousands of copies recover- able from a single DNA sample.
Exacting process The process of extracting the DNA strand is hi-tech. The protein is isolated from other body chemicals, using the centrifuging method. The DNA molecule is then cut, when it is treated with a restriction enzyme, after which the fragments are run on an electro-phoresis (EP) machine. Here, the characteristics of the DNA are exposed, and eventually become visible to the naked eye.
The process starts by mixing the tissue or samples of the blood stain recovered with the restriction enzyme (endo- nuclease) in a test tube, *'here all the proteins are extracted. The samples are embedded on a gel-plate, which has a glass base, on which some form bf starch (usually poly-acrylamide) is spread. This is then placed in a high-speed centrifuge machine.
After this, the samplol,s extracted are refrigerated. The quantity of the molecules 4 then measured in a spectro-photometer to see whether the samples are sufficient for carrying out the remaining tests. The P-CR is again carried out at this stage if larger quantities-.0f, DNA extracts are required.
The samples are the&feft in incubators overnight, after which they are mixed in high-speed rotators, to 'shake loose' the strands. The next step is electro-phoresis, in which the DNA strands (yet invisible) are smeared, at intervals, on one end of a nitro-cellular membrane (nylon sheet). This sheet is usually between 10-20 inches long, though the exact length for each test is dictated by the its protocol (the precision parameters of the test). Placed on a neutral medium, the sheet is connected to an electrical circuit vertically. The end of the sheet, where the DNA strands are smeared, are near the end of the circuit, as the DNA molecules are negatively charged. When the electrical circuit is switched on, the DNA molecules'run' in straight lines toward the ositive end of the EP tank. This process takes between 4-5 hours.
Changing the flow of the current, from top-to-bottorn to back-to-front, the traces of the path taken by the molecules is etched on the membrane. But here too, the path is invisible to the naked-eye.
The DNA strand has a certain repeating base sequence. Scientists have established that there are 4 combinations in a DNA strand. These have been termed A, T, c and G. Further, A and T form one set of combinations, and c and G the other. While the first of either set can exchange places with the second of the same set, they cannot interchange with the other set. Thus, the combination can read A-T, &-G, G-c, T-A, c-c, c-G, A-T, etc, but not A-G, G-A, C-A, T-G.
To establish the location of these combinations on the membrane, a radio labelled probe is used, which is complimentary to this sequence (A and T,, and C' and G). When this probe goes over the membrane it', locates these sequences, binding them together. But the DNA molecules, now resembling bands, still remain invisible.
The membrane is then water washed and placed on a x-ray sheet, after which it is exposed in a darkroom for a duration ranging from a few hours to a few days, depending on the protocol. At the end of this, the DNA bars are visible on the x-ray sheet. The entire process takes 20 days.
Once this is complete, the combinations are compared with the DNA profile results of the parents. The same procedure is repeated for the DNA profiling of the parents, with which it must match to confirm the identity of the body. That closes the chapter for the lab-based bio-sleuths, working in tandem with the legal investigating agencies.
IDNA profiling was preceded by various other methods of identifiption. One of them was through morphological, physical and chemical studies of available hair samples. The hair of the deceased is compared with samples retrieved by matching trace element profiles.
In the eventuality of mere fragments of tissue being available (as could be the case in a severe crash), identifying the genetic markers by fixing of blood groups and examining blood and protein systems could lead to the confirmation of the identity of a body.
The chances of error in such tests are put at 1-in-30 million cases. The test of white blood cells, or the human leucocyte antigen (HLA), is said to have an even higher rate of accuracy. The chances of duplication are 1-in-420 minion cases. However, what the scientific community and forensic experts are now pushing as the infallible.test is DNA profiling, which has a 1-in-75 billion error probability ratio.