DNA fingerprinting

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DNA fingerprinting

  1. 1. Advanced Level Teacher’s Seminar-2006 DNA Finger printing and Related techniques S.Santharooban 1
  2. 2. DNA Fingerprinting or DNA Typing• Method to identify the DNA of an individual• DNA is unique to a person, no two individuals have identical DNA except identical twins or clones• All the cells of a person has same DNA• A specimen for DNA fingerprinting can be any type of cell – Blood, saliva, sperm, muscle, teeth, bones or any DNA bearing cell 2
  3. 3. Importance of VNTRs• DNA contain exons and introns. • Exons that contain genetic information. • Introns supply no relevant genetic information at all• Introns contain repeated sequences of base pairs.• These sequences is called Variable Number Tandem Repeats (VNTR). 3
  4. 4. • VNTRs contain anywhere from 20 to 100 base pairs.• Every human being has some VNTRs.• To determine if a person has a particular VNTR, a Southern Blot is performed.• In this technique, VNTRs is probed, through a hybridization reaction, with a radioactive version of the VNTR in question.• The pattern which results from this process is often referred to as a DNA fingerprint. 4
  5. 5. Southern Blot techniqueinvolves… Invented by Prof. Ed. Southern.• Extract DNA from the sample of an individual• Digest into small fragments using restriction enzyme• Separate DNA fragments by gel electrophoresis.• Denature the DNA, • the entire DNA is changed as single-stranded.5. Apply a sheet of nitrocellulose paper to the gel (containing DNA).6. Make to permanently attach the DNA to the sheet.7. The Southern Blot is now ready to be analyzed. 5
  6. 6. • Apply a radioactive genetic probe/ a radioactive version of the VNTR to nitrocellulose filter paper.• Radioactive probe bind to target sequence/VNTRs.• Washed out excess probe.• Determine the position of the bounded probe by autoradiography. » By an X-ray filming » areas where the radioactive probe binds will show up on the film. 6
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  8. 8. Bands resulted from electrophoresis Specific probe Original sample of DNA Final bands resulted from Southern blottingPCR can also be used in the DNA fingerprinting techniques. In case,where the DNA sample in question, is too small, then the PCRtechniques is used to amplify the DNA present in it. Discussed later 8
  9. 9. Use of DNA fingerprinting• Criminal Identification and Forensics. » used in forensic medicine-confirm the suspect• Determine family relationships » paternity confirmation, family diseases• Applicable to both plants and animals 9
  10. 10. Polymerase Chain Reaction (PCR)• Invented by Kary. B. Mullis.• used to amplify specific regions of a DNA strand.• sample is crushed and added different buffers solution and primers.• Then it is put into Thermocycler to amplify the DNA strands using appropriate temperature.• The following reaction take place in the amplification. 10
  11. 11. Single strand formation at 94-980C Annealing at 680C with primers Elongation at 720C (P= Polymerase) First cycle complete. The two resulting DNA strands make up the templates for the next cycle. Thus, amount o DNA duplicated in each cycle. 11
  12. 12. What will happen to double stranded DNA if the temperature is raised to 900C???? 12
  13. 13. Epindoff tube Thermocycler 13
  14. 14. After amplification you can get a DNA strands like this 14
  15. 15. • Then Ethidium bromide is added to stain the DNA.• poured onto the electrophoresis gel.• allowed to run nearly for 1 hrs.• It results in series of bands. • Bands are laid down based on the base pairs of each type of DNA. • Bands present in the distal end of application consist the DNA of less base pairs. • Bands present in the proximal end of application consist the DNA of higher base pairs. • In between are moderate base pairs.• Obtained bands will be compared to references banding pattern. 15
  16. 16. Pouring of stained DNA samples into electrophoresis gel 16
  17. 17. Electrophoresis Apparatus 17
  18. 18. Resulted PCR bands• Bp is the base pairs of each type of DNA in a cell of interest 18
  19. 19. This is a -80 degree freezer.DNA can be stored for a verylong time in a good moistureproof deep freeze scientificfreezer. 19
  20. 20. C. S. Of Toad Stomach 20
  21. 21. T.S. Through the Toad stomach 21
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