DNA TYPING

(previously referred to as DNA fingerprinting or Genetic typing) is probably one of the most important advances in the field of forensic sciences in recent years. The first application of DNA typing in forensic science was by Dr. Alec Jeffries (Leicester University) who was called in by police to apply his new technique of DNA fingerprinting to help solve two murders in Leicestershire.

Principles of DNA typing

Genes and chromosomes A chromosome contains two complementary stands of DNA. These are long polymers of nucleic acids (nucleotides) each consisting of 1;phosphate 2; Deoxyribose 3;One of four bases which consist of adenine, thymine, guanine and cytosine (A, T, C and G). These always form base pairs based on hydrogen bounds between complementary bases. Genes are the parts of chromosomes containing stretches of DNA which actually code for proteins. A gene could be thought of as a (discrete unit of information influencing inherited characteristics.)

The scientific basis of DNA typing

The cell, the nucleus houses the DNA that codes for genetic information responsible for all cellular processes. Several DNA molecules comprise genes which in turn are located in minute bodies call chromosomes. Chromosomal regions with short tandem repeating DNA units (known as short tandem repeats or STRs) have been used as markers for human identification in forensic casework.

 DNA can also be found in another part of the cell called the mitochondrion. Unlike unclear DNA with two copies per cell, multiple copies of mitochondrial DNA (up to 100,000 copies) are present per human cell. Because of this, mitochondrial DNA analysis is the method of choice when delaying with environmentally challenged samples, e.g. identification of mass disaster victims, exhumed human remains, etc . there are at least five different forensic techniques of DNA typing. RFLP--- Restriction Fragment Length Polymorphism, the oldest technique, essentially involves radioactive fragmentation and examiner comparison PCR--- Polymerase Chain Reaction, a copying technique for smaller broken pieces of DNA, which is amplified, not cloned, and a computer or operator estimates match probabilities.

 STR---Short Tandem Repeats, a method which uses markers for short, repeating segments of micro variant allele patterns, as short as three to seven base pairs, usually involving computer expert systems although visual detection is possible. Mitochondrial DNA---- A type of PCR used on samples subjected to extreme environmental conditions, and since mitochondrial DNA is inherited solely from the mother, it has also been used in cases of disputed maternity. Rapid DNA ID Microchip-Based Genetic Detectorsthese are field-ready laptop analysis units capable of being used at crime scenes, displaying profiles onsite, or electronically uploading to a CODIS database. The technology uses the same microchips that detect genetic diseases, but modified to transport, concentrate, and hybridize DNA, and to discriminate individual genetic markers.

 RELP DNA Typing: the first step in RFLP DNA typing is extraction of the DNA from the sample, be it blood, saliva, semen or some other biological sample. The purified DNA is then cut into fragments by restriction enzymes. Take the pattern GCGC, and imagine it occurs more than one in the DNA. Application of DNA typing Paternity disputes Maternity resolution Establishment of biological relationship for immigration purpose, kidney transplantations etc. Detection of cases of child swapping Identification of rapist in rape cases including gang rape cases. Identification of mutilated remains, as in cases of murder, bomb blasts, mass disasters Identification of bodies in exhumation cases All cases of wildlife identification

METHODOLOGY COLLECTION AND FORWARDING OF FORENSIC SAMPLES FOR DNA TPING The prerequisites for successful DNA typing is an efficient method of collection of these samples from the scene of crime, and submitting them (along with control samples), to the DNA typing laboratory with minimum delay. The infallibility of DNA analysis depends mostly on the skill, ability and integrity shown by police officers and medical officers. Since DNA typing is highly sophisticated and the results are unassailable, the scientists (DNA) analysts) should be provided with the samples in the prescribed manner, so that they can process the samples and give their opinion based on the results they obtain, in the most befitting manner.

 Liquid blood samples In cases where blood samples have to be sent as evidence samples (e.g. paternity or maternity disputes,) or as control samples (e.g. criminal cases), 2-5 ml of intravenously drawn blood should be collected into sterile, leak proof screw capped tubes containing heparin or EDTA as an anticoagulant. The sample should be mixed thoroughly, but slowly, with the anticoagulant, and then the tube should be placed in a container filled with ice (e.ge. a thermos flask) if it is to be transported immediately. The sample tube should be labeled properly, displaying clearly the source of the blood sample, the name of the doctor collecting the blood sample, and date of collection, and other details such as the forwarding authority, etc.

It there is going to be some delay before the sample can be transported; it should be stored in a refrigerator. In case it appears to the medical officer who has carried out the blood collection that the person is suffering from an apparent genetic disorder, this fact should be mentioned in the forwarding note/letter of advice. This would help the DNA analyst to interpret the results correctly. And identification card should be completed and sent along with each blood sample.

 Semen samples / vaginal swabs

1. In criminal cases involving rape, or rape murder, DNA typing can conclusively help in the identification of the culprit, provided suspects/accused persons are available for obtaining their blood samples for subsequent comparison with that of the DNA profile of the seminal stain/swabs (control sample). 2. Limitations of swabs/samples. In rape cases, the role of the police officer is of utmost importance because any delay in getting the medical examination of the victim done could lead to loss of valuable evidentiary material, i.e., the seminal fluid/stain. 3. The investigating officer should get the medical examination of the victim done by a medical officer without unnecessary delay.

4. Sterile cotton ear buds can be used as swabs. 5. After collection, the swabs should be completely airdried, placed in dry sterile tubes, sealed, and labelled. Other relevant information about the sample should be enclosed separately, and sent along with the sample 6. In gang rape cases, more than two vaginal swabs should be collected and sent in separate individual tubes. The genital parts of the victim should be swabbed with wet cotton swabs. These tubes should be packed in separate vials and sent to the DNA laboratory 7. The clothes worn by the victim at the time of the offence also should be air-dried, packed in dry paper, and sent to the laboratory.

 Collection of stains from automobile/scene of crime/large material objects Body fluid stains on automobiles or large objects could be swabbed with cotton buds moistened with sterile water. Ampoules containing water for injection could be used for moistening. The cotton buds should be air-dried, placed in a clean bottle, and sent to the DNA laboratory at room temperature.

 Collection of blood/semen from individuals under field conditions Blood/semen from individuals should be collected into a sterile tube or container, and the same should be spread on a sterile bandage cloth folded several times so as to make it absorb all the fluid. This should be air dried under shade and not under direct sunlight. After complete drying, it should be placed in a clean envelope, sealed, labelled, and sent to the DNA laboratory at room temperature.

 Collection and forwarding of visceral samples Cases occur wherein mutilated bodies are recovered whose identification is difficult to establish by conventional forensic methods. In such cases, DNA typing can help to establish the source of the mutilated body with relative or absolute certainty.

 Collection and forwarding of bones, teeth, and hair 1. DNA typing can help to establish the identity of mutilated bodies, exhumed bodies, and skeletal remains with a remarkable degree of certainty. Bones, especially long and intact bones such as humerus or femur, are most suitable for DNA typing analysis. In criminal cases where skulls are recovered (with teeth), molar teeth should be detached from the upper and lower jaw bones and sent to the laboratory for analysis. If molar teeth are not available, any other tooth present could be sent for analysis.

2. Bones should be packed in clean paper/cloth and placed in a polyethylene cover, sealed, and sent to the laboratory at room temperature. Ice or preservatives need not be used for transportation of bones. Also, there is no necessity to wash the bones before they are packed. 3. Teeth should be placed in a clean polyethylene cover and sent to the laboratory for analysis. Teeth along with the skull, which have been subjected to superimposition test, are not useful for DNA analysis. Teeth should be removed from the upper and lower jaws of the skull before they are subjected to superimposition test.

4. Hair samples should be packed in clean paper sealed, and sent at room temperature. If possible hair with roots should be sent for analysis. No preservatives should be used for storing hair. At autopsy, the medical officer should always look for loose hair in the palms of a murdered person; which in some cases could be useful for identification of the culprit.

 Fingernail scrapings Fingernail scrapings in cases of rape or murder constitute a very good source of DNA of the assailant. Therefore caution should be exercised while collecting these scrapings at the time of autopsy. The palms of the victim should be placed over a clean polyethylene sheet and examined. Using a sterile sewing needle or a toothpick, the inner portions of the fingernails must be scraped gently. All the material that falls on to the polyethylene sheet should be collected and sent to the laboratory without any preservative.

 Authentication and Forwarding Blood samples should not be collected from persons who have undergone blood transfusion within three months preceding the date of collection.

 Status of DNA typing as a forensic tool Advantages It is the most conclusive method of identification of individuals (along with digital finger prints) Since DNA is very stable, the technique can be applied even on very old (aged) stains or specimens. An extremely small quantity of body fluid or tissue is sufficient for analysis. Dis-advantages DNA typing cannot differentiate between identical twins. It is very expensive Interpretation of results requires a great deal of expertise and experience.