DNA FINGERPRINTING: Synonyms: DNA Typing

DNA FINGERPRINTING: Synonyms: DNA Typing, DNA Analysis, DNA Profiling, DNA Studies, Genetic Typing, DNA Testing, Genetic Fingerprinting DNA Fingerprinting is the technique applied to prepare a profile of an individual containing DNA [ Deoxyribo Nucleic Acid] in their cell nucleus. More specifically this technique is applied to human beings. Although DNA fingerprinting is also done in Bacteria, Fungus, plants etc. In this chapter we are concerned with only Human DNA Fingerprinting. In a very simple term DNA Fingerprinting is taking photograph or auto-radiograph of DNA which is very similar to that of taking photograph of Human Being but since DNA is a very small molecule so we need some sophisticated technique to do so that is DNA Fingerprinting. The photograph/ auto-radiograph is taken in Ultraviolet light with specific DNA sequence emitting radioactive waves by a radioactive Dye. The Photograph so produced is in the form of Light and Dark Band and sequence of bands will tell us about the genetic make of a particular repeated sequence. DNA Fingerprinting is a technique employed to assist in the identification of individuals by their respective DNA profiles. DNA profiling technique was first reported in 1985 by Professor Alec Jeffreys at the University of Leicester in England

Introduction to DNA: Deoxyribo-Nucleic acid DNA is a long chain of base pairs (Adenine, Thiamine, Cytosine and Guanine) and two chains of base pairs joins with each other in a reverse pattern to form complete DNA molecule. These double helical chains are attached on histone protein to form a chromosome responsible for the characteristic features in living organism. Gene is a small portion of this DNA responsible for a particular character or trait or may responsible to code for a particular protein. Adenine and Thiamine connect with each other by two bond while cytosine and Guanine bonded with each other with three bonds. A-C-C-T-G-A-T-A-G-G-T-C-T-A-A The opposite base pair strands will be T-G-G-A-C-T-A-T-C-C-A-G-A-T-T DNA strands are read in a particular direction from the top to bottom ( 5' or "five prime" end to 3' or "three prime" end) as seen below: 5' A-C-C-T-G-A-T-A-G-G-T-C-T-A-A 3' 3' T-G-G-A-C-T-A-T-C-C-A-G-A-T-T 5' The chemical structure of DNA is the same for all organism containing DNA. The difference between organisms is the sequence of the base pairs.

Constituents of DNA  Phosphates  Deoxyribose Suger  Organic Nitrogenous Base :  Adenine  Thymine  Guanine  Cytosine  Single DNA molecule consist of 50-500 million base pairs

Chromosome: A rod-like structure of tightly coiled DNA, on histone proteins, found in the cell nucleus of plants and animals. In Human Beings, there are 23 pairs of Chromosomes 22 pairs are common in both male and female 23rd pair is different for both sexes and is responsible for the same. Restriction enzyme: An enzyme which cuts DNA at specific sites always acts upon DNA in the same manner. Broadly the procedure included for DNA Fingerprinting is  TAKE BIOLOGICAL SAMPLE  EXTRACT WHOLE DNA  CUT DNA IN SMALL PIECES  RUN ON GEL  TAKE PHOTOGRAPH AT DIFFERENT TIME

Methods of DNA Fingerprinting for forensic importance Methods applied in DNA Fingerprinting: 1. RFLP Method or Restriction Fragments lengh polymorphism: In this method biological samples like blood, semen, tissues, muscles, bones etc are collected and DNA is extracted with the help of standard phenol-chloroform method or may be with the help of commercially available DNA Extraction Kits. The Extracted DNA is mixed with restriction enzymes which breaks the DNA into smaller pieces. The Fragmented DNA is run on gel electrophoresis under the influence of electric field. The Smaller fragments moves faster as compared to large fragments. The distribution of DNA band pattern is transferred to a nylon sheet by placing the sheet on the gel and soaking them overnight by a technique known as SOUTHERN BLOTTING . A radioactive DNA probe is prepared which binds with specific DNA sequences to produce a fluorescent image. It is known as hybridization. Hybridized probe fragments are visualized by auto-radiography. The bands on the X-Ray film appears corresponding to hybridized fragments. This is DNA Fingerprint. 2. VNTR and STR: Variable number tandem repeats, represent specific locations on a chromosome in which tandem repeats of 9-80 or more bases repeat a different number of times between individuals. These regions of DNA are readily analyzed using the RFLP approach and a probe specific to a VNTR locus. The fragments are a little shorter than RFLPs (about 1-2 kilo base pairs), but are created through the exact same process. Unlike VNTRs which analyze minisatellites that have repeat sequences of 9-80 base pairs, STRs use microsatellites which have repeat sequences of only 2-5 base pairs, introducing the “less is more” philosophy to the world of DNA fingerprinting. This was a big step forward in forensic science since the length of DNA fragment being analyzed is short enough to be amplified by polymerase chain reaction. 3. Polymerase Chain Reaction: PCR is done when the amount of recoverable DNA is small. The Smaller amount of DNA is amplified with the process of PCR. It is like doing photocopy of a small DNA fragments. In this method the two strands of DNA are separated and allowed to regrow their own fragment under controlled tempeture and availability of raw material [OLIGONUCLEOTIDE PRIMERS, DEOXYNUCLEOTIDE ] and catalysts [Magnesium Chloride].

USES:IDENTIFICATION          

Mass Disaster cases including several casualties which need to be identified as separate entity. Bomb Blast Cases Fragment Remains Unidentified bodies or Mutilated Remains Mass Casualties: Train and Air Accidents Doubtful Identification cases Missing Soldiers Missing Foreigners Wildlife identification Exhumation Cases

USES: PARENTHOOD  Disputed Maternity  Disputed Paternity  Exchange of Child in hospital  Exchange of child in early stage  Founding Missing child after long time.  Transfer of Property cases.  Citizenship Disputes  Rape cases USES: OTHERS  Diagnosis of inherited disorders.  Biological relationship for transplant  Detection of Bacteria and other organisms.  Hit and Run Cases of Road Traffic Accidents.  Disputed Diagnosis in case of histopathological examination an doubtful cases relating patient with particular sample. Collection of Samples for DNA Extraction  BLOOD  Whole Liquid Blood: About 5ml of whole liquid blood is taken in preferably EDTA vial.  Blood Clot : whole blood clot is taken if size is small.  Wet Blood Stains: Blood is taken on sterile cotton swab and air dried and then sent to Lab in zip-locked polybags.  Dried Blood Stains: whole dried blood is sealed in zip-locked polybag.  SEMEN  Dried seminal stain similarly as blood  Wet seminal stains  Hair Root.  Saliva and Salivary Stains taken in glass tube  Body tissue cells: taken in clean sterile container.  Fingernail Scraping taken in polybag.  Postmortem samples like Muscle, Spleen, Bone marrow, organs, Vascular pulp of tooth are taken in clean sterile plastic tube without any preservative. If delay is there then freeze at – 20 C

Paternity Disputes  VNTR patterns can be used to establish paternity and maternity. The patterns are so specific that a parental VNTR pattern can be reconstructed even if only the children's VNTR patterns are known  Parent-child VNTR pattern analysis has been used to solve standard fatheridentification cases as well as more complicated cases of confirming legal nationality and, in instances of adoption, biological parenthood.

Advantages of DNA Fingerprinting  DNA id stable molecule  Small Quantity of Sample is needed  Reliable technique  Permanent record .  Easy availability of DNA samples  Repetition of same results with same or different samples.  Lack of knowledge of Murdered/culprit. Disadvantages  Identical Twins  Expensive and Sophisticated method  Expert knowledge is needed  Intermixing of samples of culprit with others available at crime scene.  Lack of police training in sample collection.  Require more time and hence accused can run away.  Chances of error are there.  Lack of availability of DNA Laboratories.  Chances of deliberate samples exchanges.  Decomposition of Samples adversely effect results

References: 1. Im ran Sa b ri , S h az i a Im r an , F o r en si c D N A Is ol ati on an d R e c o v er y i n Bl o od an d S em en . L amb e rt s A cad e mi c P u bl i sh er , G e rman y . ISB N: 9783 843 3810 31 . 2. S ab r i I., Us man i J. A. , H an i f S. A. , K h an A.U . : E ff e ct o f te m pe r atu r e an d ag ei n g on r ec o v e ry of DN A i n fo r en si c bl ood sa mpl e s. In d i an In t e rn et J. F o r en si c . M ed . & T ox . July- Sept. 2 008 ; 6[3 ]:69 - 77 . 3. P V Guharaj, M R Chandran: Forensic Medicine, Second Edition. Orient Longman. ISBN 139788125024880. 4. Kate Brinton, Kim-An Lieberman. Basics of DNA Fingerprinting: http://protist.biology.washington.edu/fingerprint/dnaintro.html 5. Reddy KSN: Essentials of Forensic Medicine and Toxicology: Publisher: K. Suguna Devi. 6. Ballantyne, John, George Sensabaugh, and Jan Witkowski. DNA technology and forensic science. Cold Spring Harbor, NY: Cold Spring Harbor Lab, 1989. 7. Jeffreys AJ, Wilson V, Thein SL.Hypervariable 'minisatellite' regions in human DNA.Nature. 1985 Mar 7-13;314(6006):67-73. 8. Betsch, David (2005) DNA Fingerprinting in Human Health and Society. http://www.extension.iastate.edu/Publications/NCR550.pdf. 9. Christopher Lach, Thomas Patsis: DNA Fingerprinting: An Interactive Qualifying Project Report http://www.wpi.edu/Pubs/E-project/Available/E-project-011306130417/unrestricted/IQP.pdf