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1. DNA PROFILING Presented by, Ijaj Ahmed 3 rd BAMS GAMC,Mysore . Guided by, Dr.Adarsh sir

2. Contents 1. Introduction 2. Definition of DNA Profiling 3. Methods of DNA PROFILING 4. Explanation of procedure 5. Medico-legal importance

3. Introduction DNA profiling is one of the late 20 th century’s discovery, has revolutionized forensic investigations.
The Eureka shout shook England again and was heard around the world when roughly 100 years later ALEC JEFFREYS at the University of Leicester, in UK, found extraordinarily variable and heritable patterns from repetitive DNA analyzed with multi-locus probes. He called the method’ DNA FINGERPRINTING.

4. The chances that DNA profiles in two individuals aresimilar are about 1 in 30 billion to 300 billion Le half the population of the world. The techniques that are applied in identity testing areDNA fingerprinting, DNA profiling, and DNA typing. Although there are some technical differences between these tests, the terms have been used interchangeably.

5. BEFORE DIVING INTO DNA PROFILING TOPIC WHAT IS DNA ? Deoxyribonucleic Acid (DNA) DNA is an acronym, which stands for deoxyribonucleic acid. Every cell in an individual’s body, with the exception of red blood cells and eggs or sperm, contains the full genetic program for that individual in its DNA . The program is coded by four chemical compounds called, bases, or subunits - Guanine, Cytosine, Adenine and Thymine (usual y abbreviated as G, C, A and T),that are arranged into extremely long sequences. Groups of three bases (known as codons) code for the 20 amino acids, the basic building blocks of life. The amino acids in turn are linked together to form proteins. There are also stop codons signaling termination of the amino acid sequence. Though the code is well understood, biologists are still a long way from understanding how the code is expressed; for example, although each cell in an individual contains identical genetic information, the way the information is expressed in a liver cell is very different from the way it is expressed in a brain cell.

6. The human genome which consists of about 3 billion base pairs harbours genetically relevant information which is essential for the characterization of each individual. It is believed that genetically relevant information represents less than 10 % of the human genome. This minor part of the gene-coding DNA has been subjected to evolutionary pressure and selection mechanisms ensuring the development of higher organized organisms. The other 90% of the genome is junk DNA, a term which is more of a misnomer since their functions are still unknown rather than useless. A part of this non-coding DNA is comprised of repetitive sequences. Highly polymorphic spots in these non-coding regions are referred to as mini- or micro-satellites characterized by repeated blocks of DNA. The single-locus satellites are localized at a specific site of a given human chromosome, while multi -locus satellite elements or short tandem repeats (STRs) are spread throughout the entire genome.

7. There exists a significant level of diversity within the genome. During evolution, the process of selection involves non-directed mutations, which may be maintained when generation of a neutral or improved ability is successful while negative mutations normally get lost. The non-coding regions of the human genome are not regulated by these rules of selection and maintenance as long as they are not affecting the survival capacities of the individual. This is the reason for the accumulation of mutations leading to the generation of genetic diversity within the non-coding genomic DNA. Exceptions are polymorphisms in gene-coding regions, which reveal a high genetic stability combined with a very low mutation frequency.DNA is a sturdy molecule which can tolerate wide range of temperature, pH and other factors. DNA mixed with detergents, oil, gasoline and other adulterants does not alter its typing characteristics

8. DNA FINGERPRINTING DNA fingerprinting( DNA typing, DNA identification, DNA profiling or genetic typing) is a technique that is capable of distinguishing every individual with the exception of identical twins and clones. It depends on the fact that no two people have exactly the same DNA sequence (with the exception mentioned) and that although only limited segments of a person's DNA are scrutinized in the procedure, those segments will be statistically unique. Consequently, DNA fingerprinting is rapidly becoming the primary method for identifying and distinguishing among individual human beings.

9. The main difference between DNA fingerprinting and DNA profiling is that DNA fingerprinting is a molecular genetic method that allows the identification of individuals according to the unique patterns of DNA, whereas DNA profiling is a forensic technique used in both criminal investigations and parentage testing.

10. There exists a significant level of diversity within the genome. During evolution, the process of selection involves non-directed mutations, which may be maintained when generation of a neutral or improved ability is successful while negative mutations normally get lost. The non-coding regions of the human genome are not regulated by these rules of selection and maintenance as long as they are not affecting the survival capacities of the individual. This is the reason for the accumulation of mutations leading to the generation of genetic diversity within the non-coding genomic DNA. Exceptions are polymorphisms in gene-coding regions, which reveal a high genetic stability combined with a very low mutation frequency DNA is a sturdy molecule which can tolerate wide range of temperature, pH and other factors. DNA mixed with detergents, oil, gasoline and other adulterants does not alter its typing characteristics.

11. Two methods of DNA analysis are in common use: i.RFLP (restriction fragment length polymorphism) ii. PCR (polymerase chain reaction)

12. Restriction Fragment Length Polymorphism (RFLP) is a difference in homolog sequences that can be detected by the presence of fragments of different leng 8/25 digestion of the DNA samples in question with specific restriction endonuclease as a molecular marker, is specific to a single clone/restriction enzyme combination. Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and highly locus-specific. An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus. Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP probes. The RFLP probes are frequently used in genome mapping and in variation analysis (genotyping, forensics, paternity tests, hereditary disease diagnostics, etc.). RFLP

13. HOW IT WORKS? In the human genome, in between the active base pairs which code for a particular protein, there is large number of inactive base pairs forming 95% of DNA, which is considered as junk DNA' or 'filler DNA' or 'nonsense DNA. Technically, these introns' separate the exons' which serve as protein patterns. DNA fingerprinters overlook the DNA in genes, in favor of junk DNA' between the genes.In junk DNA short sequences of base repeat themselves over again like a stutter (repetitive DNA), e.g. CGTA, CGTA, GACA, GACA, etc. The regions containing repetitive DNA demonstrating hypervariability from person to person are called satellite DNA' which shows an extremely high degree of variability, and these variants are called 'variable number tandem repeats' (VNTR) or ' minisatellites . Selected regions of VNTR are broken into fragments using special enzymes (restriction endonucleases). The resulting fragments are called restriction fragments length polymorphisms (RFLP). Gel electrophoresis can be used to separate and determine the size of the RFLPs (fragments are of variable lengths). The exact number and size of fragments produced by a specific restriction enzyme digestion varies from individual to

14. PROCEDURE : The most common method of DNA typing is RFLP analysis of VNTR loci. Isolation/extraction of DNA : DNA must be recovered from the cells or tissues of the body. Only a small amount of tissue-blood, hair or skin-is needed. For example, theamount of DNA found at the root of one hair is usually sufficient. 2. Cutting and sizing: Special enzymes calle d restriction enzymes are used to cut the DNA a t specific places. For example, an enzyme called EcoR1, found in bacteria, will cut DNA only when the sequence GAATTC occurs. 3. Sorting by gel electrophoresis : The DNA pieces are sorted according to size by a sieving technique called electrophoresis. The DNA pieces are passed through an agarose gel. This results in separation of the DNA fragments based on their length (size).

15. 4 .Transfer of DNA to nylon (Southern blotting) : It is possible to identify specific DNAfragments that hybridize with a complimentary genetic probe. However, it is impossible to hybridize a probe to DNA fragments contained in a gel. For this reason, the DNA is usually denatured and then transferred to a nitrocellulose or nylon membrane which picks up the DNA like a blotter picks up ink. DNA is transferred to the membrane by capillary actionand fixed bybaking , making it accessible to a probe. The resulting blot formed is essentially a replica of the gel. 5. Hybridization: Adding known radioactive DNA probes (short sequence probe, complimentary to the region of DNA which one wishes to detect) to the nylon sheet leads to fragment location. The nylon membrane is immersed in a solution that contains DNA probe impregnated with radioactive P 32. Each probe typically sticks in only one or two specific/complementary sequences on the nylonsheet . This process is termed as hybridization. 6. Washing: T he membrane is washed to remove excessor unbound probe and exposed to an X-ray film. The resulting spots on the X-ray film correspond to the locations of the fragments in thegel that are complimentary to the probe (autoradiography).Nowadays, many radioactive probes are detected by chemical luminescence which is analyzed by computer scanners, eliminating the need for autoradiography. 7. DNA fingerprint: The final print is known as an autoradiograph or 'DNA fingerprint'which appears as lines on the film.

16. PCR PCR is a technique used for amplifying sample of DNA fragments in vitro. -PCR won its discoverer Kary B. Mullis, a Nobel Prize in chemistry for his work in 1993. In this process, a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzable sample of a piece of DNA; the process is also called DNA amplification. -PCR itself does not accomplish DNA typing, but increases the amount of DNA availablefor typing. It is used to produce multiple copies of segments from a very limited amount of DNA Once a sufficient sample has been produced, the pattern of the alleles from a limited number of genes is compared with the pattem from the reference sample. A nonmatch conclusively excludes a suspect, but the technique provides less certaintywhen a match occurs.

17. Procedure The theory behind PCR is based on certain aspects of DNA replication. The enzyme DNA polymerase helps to expand a short sequence into a longer one or a polymer. But DNA polymerase needs single stranded DNA that acts as a template for the synthesis of a new strand. It also requires a small portion of double stranded DNA to initiate synthesis (primers). Then, new DNA strands are synthesized and amplified behind the primer

18. Three steps are involved in this process : Denaturation : Heating the double stranded DNA to almost boiling will dissociate andbecome single stranded. 2. Annealing : Cooling the reaction will cause the primers to pair up with the single- stranded template (annealing). On the small length of double-stranded DNA. 3. Extension : DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule.primer and template), the polymerase attaches and starts copying the template. Each separated strand can serve as a template for synthesis as long as primer is provided for each strand, and the reaction is cooled to cause the primers to bind. The primers are chosen to flank the region of DNA that is to be amplified. New primer bindingsites are generated on each synthesized DNA strand. This cycle of DNA denaturing, primer annealing and strand synthesis is repeatedmultiple times, thereby amplification of the target DNA. After 20 heating and cooling cycles, this exponential process yields 220, or more than a million copies of the target sequence. The process is completely automated with thermocyclers that contain a heating blockand microprocessors. The time and temperature can be programmed for repetitive cyclesof heating and cooling, alleviating manual intervention.

19. Criteria to determine the source: DNA testing laboratories use a two-step process to determine if two samples arose from one source. First, DNA-banding patterns are compared visually. If banding patterns of a sample in question do not match a known DNA sample, exclusion is declared and no further analysis is required. Second, a visualized match is verified by a technique called computer assisted allele sizing which is done by computer software. (Basically, the calculated sizes of an apparent match should fall within 2.5% of each other. When samples fall outside of the 2.5% window, they should be considered ‘nonmatching.’)( If the DNA-banding pattern of a sample cannot be positively determined du e to tech nical problems, the results should be considered ‘inconclusive.’ )

20. Samples Col l ected from Living Subjects i . Blood (most common sample) ii. Buccal epithelial cells (buccal swabs) iii. Hair follicles with roots (plucked hair)

21. Samples Col l ected from Dead Bodies DNA can be isolated and tested from practically any postmortem tissue, although after death it will undergo progressive degradation. DNA is broken down into fragments by autolytic and bacterial enzymes, especially DNases. i . In relatively fresh dead bodies, unclot ed 10 ml of blood (EDTA anticoagulated in a sterile tube) is the preferable source of DNA.10,11 Buffers (e.g. those containing EDTA) are designed to inhibit the activity of nucleases that can breakdown DNA. Due to settling out of WBCs, clotted blood is not a good source of DNA.12 ii. Brain tissue is a good source of DNA in intermediate postmortem intervals iii. Hard tissue (bone and vascular pulp of teeth) is the best source of DNA in cases of advanced decomposition. Best material is said to be muscle or spleen if decomposition is establishing; bone mar ow (from femur) and teeth (usual y molars) are also recommended. 10 ml of blood or some tissue or swabs in a sterile tube should be taken and frozen at –20°C, if there is likely to be some delay in transmission to the laboratory.

22. Samples Encountered in Forensic Practice Blood (EDTA/heparinized/clotted/stain on cloth,newspaper , wood or tiles) • Semen (stain on cloth/paper/floor) • Hair (head/body/pubic) • Tissue (bone marrow/muscle/spleen/fingernail scrapings) • Mouth swabs and saliva stain on cigarette buds/ licked envelope

23. How to store DNA ? DNA is very sensitive and can easily degrade in certain conditions. Thus, proper storage is required to ensure high experimental standards. There are several mechanisms to store DNA for long periods of time. Storage in vitreous state Medium length storage Repeatedly freezing and thawing DNA

24. Medico legal importance. Identification: It is used to link suspects to biological evidence—blood or semen stains, hair or items of clothing found at the scene of a crime. It is used to establish identity of an assailant in sexual assaults, like rape, incest and bestiality. Diagnosis of inherited d isorders in children, newborn and prenatal babies: It includes cystic fibrosis, hemophilia , Huntington’s disease, familial Alzheimer’s, sickle cell anemia and thalassemia. Genetic counselors use DNA fingerprint information to help prospective parents understand the risk of having an affected child or decisions concerning affected pregnancies. Iii. Developing cures for inherited disorders: By studying the DNA fingerprints of relative who have a history of some particular disorder or by comparing large groups of people with and without the disorder, it is possible to identify DNA patterns associated with the disease in question.

25. Establish paternity in custody and child support litigation. In these applications, DNA fingerprints bring a nearly perfect accuracy to the determination. Identifying the remains of soldiers: In US armed services, a program is there to collect DNA fingerprints from all personnel for use later, in case they are needed to identify casualties or persons missing in action. Biologists routinely use it, particularly to protect endangered species. Accidents/mass disaster investigations and postmortem identification of skeletal remains/ mutilated bodies.

26. Limitations Of DNA Testing Generally, courts have accepted the reliability of DNA testing and admitted DNA test results into evidence. But, DNA fingerprinting is controversial in a number of areas. Uniqueness of DNA fingerprinting: DNA segments rather than complete DNA strand are ‘fingerprinted’, a DNA fingerprint may not be unique. Time constraints: The process is lengthy, with each of four or five loci exposed sequentially, it usually takes 10 weeks. Invasion of privacy and ethical concerns: In the US, the FBI has created a national database of genetic information called the Combined DNA Index System (CODIS). Similar database is present in UK also. The database contains DNA obtained from convicted criminals and from evidence found at crime scenes. Some experts fear misuse of the database, such as identifying individuals with stigmatizing illnesses such as AIDS.

27. Suspects who are unable to provide their own DNA experts may not be able to adequately defend themselves against charges based on DNA evidence. Unlike fingerprints, DNA profile cannot be enlarged and shown in the court of law.

28. References Forensic application of DNA ‘ fingerprints’.Gill P, Jeffreys AJ, Werrett DJNature . 1985 Dec12-18; 318(6046):577-9. [PubMed] [Ref list] National Research Council, National Academy of Sciences.DNA Technology in Forensic Science. Washington, DC: National Academy Press; 1992. p. 156. (Cited as NRC report.) [Google Scholar] [Ref list] Biswas GautamI,Review of Forensic Medicine &Toxicology,Chapter-32 Dna Fingerprinting Edition 2/e , publishing year 2012, page number 386-391