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DNA Profiling

DNA Profiling. (DNA fingerprinting). DNA is Tightly Packaged into Chromosomes Which Reside in the Nucleus. Model of DNA DNA is Comprised of Four Base Pairs. What is DNA Profiling?. A technique used by scientists to distinguish between individuals using only samples of their DNA.

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DNA Profiling

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  1. DNA Profiling (DNA fingerprinting)

  2. DNA is Tightly Packaged into Chromosomes Which Reside in theNucleus

  3. Model of DNADNA is Comprised of Four Base Pairs

  4. What is DNA Profiling? A technique used by scientists to distinguish between individuals using only samples of their DNA

  5. Who Invented it? • The process of DNA fingerprinting was invented by Alec Jeffreys at the University of Leicester in 1985.

  6. Stages of DNA Profiling • Stage 1: Cells are broken down to release DNA If only a small amount of DNA is available it can be amplified using the polymerase chain reaction (PCR)

  7. Stages of DNA Profiling • Stage 2: The DNA is cut into fragments using restriction enzymes. Each restriction enzyme cuts DNA at a specific base sequence called the restriction site.

  8. DNA Restriction Enzymes Discovery • Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack • Endonucleases (restriction enzymes) cleave DNA strands • In simpler terms, bacteria use them to cut up the viral DNA • Over 3,000 known enzymes

  9. Enzyme Site Recognition Restriction site • Each enzyme digests (cuts) DNA at a specific sequence called a restriction site • Enzymes recognize 4- or 6- base pair, palindromic sequences (read the same both backward and forward) (eg GAATTC) Palindrome Fragment 2 Fragment 1

  10. Common Restriction Enzymes EcoRI – Eschericha coli – 5 prime overhang Pstl – Providencia stuartii – 3 prime overhang

  11. An EcoR1 restriction enzyme

  12. Restriction enzymes • Restriction endonucleases • http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio37.swf::Restriction%20Endonucleases • cleave DNA at specific points

  13. Stages of DNA Profiling • The sections of DNA that are cut out are called restriction fragments. • This yields thousands of restriction fragments of all different sizes • A tandem repeat is a short sequence of DNA that is repeated at a specific chromosomal locus (location) • These repeats are very unique to each individual

  14. Red boxes represent the repeat unit and the blue lollipops represent cut sites for a restriction endonuclease. (Here 3 different variants, may be 50 in reality).

  15. Stages of DNA Profiling • Stage 3 • Agarose gel electrophoresis. • Electrical current carries negatively-charged DNA through gel towards positive (red) electrode Buffer Dyes Agarose gel Power Supply

  16. Agarose Gel electrophoresis • DNA is placed at one end of a gel • An electrical current is applied to the gel • DNA molecules are negatively charged and move toward positive end of gel • Smaller molecules move faster than larger ones

  17. Stages of DNA Profiling DNA is separated on basis of size. Notice the shorter fragments make it through the gel quicker than the longer fragments

  18. Stages of DNA Profiling • A radioactive material is added which combines with the DNA fragments to produce a fluorescent image. • A photographic copy of the DNA bands is obtained.

  19. Stages of DNA Profiling Stage 4: • The pattern of fragment distribution is then analyzed.

  20. Analysis of Stained Gel Determining restriction fragment sizes • Create standard curve using DNA marker • Measure distance traveled by restriction fragments • Determine size of DNA fragments • Identify the related samples

  21. Molecular Weight Determination Fingerprinting Standard Curve: Semi-log Size (bp) Distance (mm) 23,000 11.0 9,400 13.0 6,500 15.0 4,400 18.0 2,300 23.0 2,000 24.0

  22. Uses of DNA Profiling • DNA profiling is used to solve crimes and medical problems

  23. Crime • The DNA profile of each individual is highly specific. • The chances of two people having exactly the same DNA profile is 30,000 million to 1 (except for identical twins).

  24. Biological materials used for DNA profiling • Blood • Hair • Saliva • Semen • Body tissue cells • DNA samples have been obtained from vaginal cells transferred to the outside of a condom during sexual intercourse.

  25. DNA Profiling can solve crimes • The pattern of the DNA profile is then compared with those of the victim and the suspect. • If the profile matches the suspect it provides strong evidence that the suspect was present at the crime scene (it does not prove they committed the crime). • If the profile doesn’t match the suspect then that suspect may be eliminated from the enquiry.

  26. Example • A violent murder occurred. • The forensics team retrieved a blood sample from the crime scene. • They prepared DNA profiles of the blood sample, the victim and a suspect as follows:

  27. Was the suspect at the crime scene? Suspects Profile Blood sample from crime scene Victims profile

  28. Famous Cases • Colin Pitchfork was the first criminal caught based on DNA fingerprinting evidence. • He was arrested in 1986 for the rape and murder of two girls and was sentenced in 1988.

  29. Did suspect 1, suspect 2, or the boyfriend leave the sperm collected as evidence? • Do the female cells found at the crime scene belong to the victim? • Note that the more markers you use to cut the DNA, the more bands you will get. This increases the ability to tell different people apart.

  30. Famous Cases • O.J. Simpson was cleared of a double murder charge in 1994 which relied heavily on DNA evidence. • This case highlighted lab difficulties.

  31. CRIME SCENE • Shown to the right are DNA "fingerprints" taken from seven (7) suspects. • The isolated column in the center is the DNA banding pattern taken from a bloodstain at the scene of a crime. • Your task is to compare the crime scene sample with the patterns of the seven suspects to determine if one of the suspects has similarity with the crime scene sample.

  32. Solving Medical Problems DNA profiles can be used to determine whether a particular person is the parent of a child. A childs paternity (father) and maternity(mother) can be determined. This information can be used in • Paternity suits • Inheritance cases • Immigration cases

  33. Example: A Paternity Test • By comparing the DNA profile of a mother and her child it is possible to identify DNA fragments in the child which are absent from the mother and must therefore have been inherited from the biological father.

  34. Is this man the father of the child? Mother Child Man

  35. Famous cases • In 2002 Elizabeth Hurley used DNA profiling to prove that Steve Bing was the father of her child Damien

  36. Shown at the right are DNA "fingerprints" from a new mother (M, Fern LaPlante), her recently born child (C), and two possible fathers (F1, Ross and F2, Rick). As is to be expected, the mother and the child share a few DNA bands since they also share 50% of their genes but they each have bands that are not represented among the bands of the other. • What do you think - was it Ross (F1) or was it Rick (F2)? Paternity

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