DNA FINGERPRINTING SMG

A brief account of DNA fingerprinting, its applications and limitations

1. DNA FINGERPRINTING Dr. Saji Mariam George Associate Professor Assumption College Autonomous Changanacherry

2. DNA FINGER PRINTING (DNA PROFILING, DNA TYPING, MOLECULAR FINGERPRINTING, GENETIC FINGERPRINTING) One of the applications of Biotechnology - in Forensics. Developed by Alec Jeffreys (1985 ).

3. DNA fingerprints are specific banding patterns on Southern blots of genomic DNA cleaved with a specific restriction enzyme and hybridized to appropriate DNA probes – Autoradiogram is taken to get a DNA print pattern of the individuals. From the DNA print pattern, the individuals are distinguished on the basis of bands in their DNA prints . An individual can be identified at molecular level.

4. Human genome - 3billion nucleotides. No two individuals have the same nucleotide sequences. The uniform nature of DNA in a single individual and the genetic variability between individuals make DNA finger printing possible.

5. DNA finger printing relies not on full genome sequencing , but on identifying differences in some specific regions in DNA called repetitive sequences ( a small stretch of DNA is repeated many times) – satellite DNA or microsatellites or minisatellites – highly variable : Variable Number Tandem Repeats(VNTRs). Copy numbers varies from chromosome to chromosome in an individual. Each organism has a unique pattern of VNTRs - can provide valuable evidence in cases of uncertain identity.

6. Variable Number Tandem Repeats(VNTRs). Image:https :// slideplayer.com / slide/14307419

7. VNTRs are very similar between closely related humans, but dissimilar between unrelated individuals. Do not code for any proteins – form a large portion of human genome. Sequences show high degree of polymorphism due to mutations and form the basis of DNA fingerprinting- Comparing a number of VNTRs in a given area, one can identify a person easily. (DNA polymorphism – an inheritable mutation in a population at high frequency).

8. Steps in DNA Fingerprinting Isolation of cell DNA.- DNA is isolated from the source materials – a drop of blood, semen, teeth, bones, hair follicle, tissues , suspected individuals etc. Digestion of DNA by restriction endonuclease – DNA is cut with a restriction enzyme that cut on either side of a minisatellite (VNTRs) .

9. 3. PCR amplification – Since samples provide a small amount of DNA, it is amplified by PCR (Polymerase Chain Reaction). 4. Separation of DNA fragments by Gel electrophoresis Each DNA restriction digest of the resource persons and the source material is poured into the wells of an electrophoresis gel and electrophoresis is carried out.

10. The minisatellites separate according to their lengths. The gel is chemically treated (alkali treatment – 0.5 M NaOH ) or heated to denature the DNA to form single strands. ssDNA (single stranded DNA) is capable of binding to single stranded probes that can be used to detect unique DNA sequences.

11. 5.Southern blotting and baking – the separated DNA fragments are transferred to a nylon membrane or a nitrocellulose filter paper by placing it over a gel. The nitrocellulose filter having DNA fragments is dried in between dry filter papers at high temperature (Baking).

12. 6. Selection of DNA probe Single stranded fragments of DNA containing the complementary code for a specific sequence of bases. The targeted area on the DNA sample is called a locus. A single locus probe targets a sequence that appears in only one position on the genome. A multi locus probe attaches to sequences in many places in the genome.

13. DNA probes developed by Alec Jeffreys are used for DNA finger printing. In India, BKm probe – developed from mini satellite DNA of Banded Krait by Dr Lalgi Singh , Centre for Cellular and Molecular Biology, Hyderabad.

14. 7. Hybridization with labelled VNTR probe (Filter Hybridization) Binding of the DNA fragment with the complementary probe - Nitrocellulose filter is placed in hybridization solution containing the radioactively labelled probe – The probe DNA binds with appropriate minisatellites and form duplex DNAs.

15. 8. Detection of hybridized DNA fragments by Autoradiography After hybridization, the filter is washed with a wash solution to remove unbound probes. An X-ray film is placed over the filter for about 3 hours – The radioactivity of the probes makes dark spots on the X-ray film – an irregular ladder of dark spots develops on the X-ray film for each DNA sample – Each ladder represents a DNA print of an individual. All DNA prints on the X-ray film together constitute a DNA finger print pattern.

16. 9. Analysis of DNA print pattern. Compare two or more autoradiographs to see the band match. Based on the degree of band match, we can arrive at a conclusion regarding the identification of an individual.

17. Applications of DNA Fingerprinting Forensic applications A powerful forensic tool - DNA finger prints(VNTR prints) were first used as evidence in criminal case in 1988. DNA from any part of the body - hair follicle, semen, a drop of blood etc. from the crime scene is enough to establish innocence or guilt.

18. Can be used to identify a criminal – a murderer, rapist etc. whose DNA may match evidence left at crime scenes. It can also be used to identify a victim in cases where the body may be disfigured and teeth or other identifying features may be destroyed.

19. An analysis of the DNA print pattern shows that the bands of the specimen and the suspect No. 1 match. Hence he is the criminal. The suspects No. 2 & 3 are not guilty . Image:https ://prezi.com/

20. To settle disputed Parentage ( Paternity or maternity tests). DNA samples are obtained from the cells of the child, the mother and possible fathers and DNA finger prints were prepared. When the DNA fingerprints were compared, all the bands in the child’s DNA print should be present in the combined DNA prints of the parents.

21. For each pair of homologous chromosomes , the child will have received one from each parent - Thus approximately, half of the bands in the child’s DNA print will result from DNA sequences inherited from the mother and the other half from the DNA sequences inherited from the father. The accuracy can be increased by using more probes – more DNA polymorphisms can be surveyed and a large proportion of the child’s DNA and parents’ DNA can be compared.

22. A critical analysis of the band match revealed that the bands of the child and the alleged father on the fingerprint pattern on the right side match – hence he is the biological father. (Paternity inclusion).

23. 3.DNA fingerprints can be used to determine genetic family relationships ( sibling relationships or other kinships) Example Daughter 1 and son 1 share RFLPs (band match ) with both Mom & Dad ( Coloured blue & yellow respectively ).(RFLP-Restriction Fragment Length Polymorphism) Daughter 2 has RFLP of Mom , but not the Dad, D2 - is the child from mothers previous marriage . Son 2 does not have RFLPs from either parent – Son 2 is adopted. Image: http ://mol-biol4masters.masters.grkraj.org/

24. 4. Personal Identification DNA fingerprints can be used to generate DNA profile of an individual. Can be used as a type of genetic barcode to identify individuals.

25. Limitations of DNA Fingerprinting 1. Requires further standardization and quality control to be accepted as a tool. 2 . There are only a few labs around the world that can give accurate results. 3 . There is 1 in 50 billion chance of two DNA sequences being similar.

26. 4. The probability of human error in producing prints and the methods for calculating the probability that two individuals have identical fingerprints – researchers must have reliable information about the frequency of the DNA polymorphism in the population in question – if inbreeding is common in the population, the probability of identical fingerprints will increase.

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