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Southern Blot

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  1. Southern Blot By: Jacqueline Jai

  2. Southern Blot • Southern Blot-a piece nitrocellulose paper containing spots of DNA ready for identification by a suitable molecular probe. • Southern Blot is a copy of DNA profile January 28, 2003

  3. Interesting Facts about DNA Analysis

  4. DNA evidence-has many uses within the legal system and criminal cases. Proving someone guilty or innocent for a crime they have or have not committed. Identification Paternity Testing DNA Evidence First criminal identification card filed by the NY State Bertillon Bureau January 28, 2003

  5. Criminal Cases • DNA evidence has exonerated people accused of committing crimes. • Only about 30% of all DNA tests run by the FBI have exonerated an accused person; DNA evidence is still not as useful as fingerprinting. January 28, 2003

  6. Identification • Used to determine the sex, race, or even name of unnamed victims of crimes. • Used in military to identify those who have died in battle, similar to the purpose of dog tags. Typical dog tags January 28, 2003

  7. Paternity Testing • Evidence can be used to compare the DNA of the suspected parent(s) and that of the child and determine the real parent. January 28, 2003

  8. DNA Profile

  9. The Basics to Creating a DNA Profile (Agenda) • Collect the DNA • Isolate the DNA • Cut DNA • Variable Number Tandem Repeats (VNTRs) • Sort DNA through Gel Electrophoresis • Transfer DNA to a solid support • Methods of Tranferring • The Hybridization Reaction • Denatured and Nicked DNA • Radioactive Probe • Continuation of the Hybridization Reaction • Comparing DNA fingerprints A DNA Profile January 28, 2003

  10. Collect the DNA

  11. Collect DNA • Collect DNA sample • Blood, hair, tissue, semen • Clean DNA • DNA found at a crime scene usu. dirty • Must be clean before analyzed A piece of DNA January 28, 2003

  12. Agenda Revisited • Collect the DNA • Isolate the DNA • Cut DNA • Variable Number Tandem Repeats (VNTRs) • Sort DNA through Gel Electrophoresis • Transfer DNA to a solid support • Methods of Transferring • The Hybridization Reaction • Denatured and Nicked DNA • Radioactive Probe • Continuation of the Hybridization Reaction • Comparing DNA fingerprints January 28, 2003

  13. Isolate the DNA

  14. Isolate DNA from the rest of cellular material in nucleus. Done chemically or mechanically. Chemically Use detergent to wash extra material from DNA Mechanically Apply large amounts of pressure to “squeeze” out DNA Isolate the DNA January 28, 2003

  15. Agenda Revisited • Collect the DNA • Isolate the DNA • Cut DNA • Variable Number Tandem Repeats (VNTRs) • Sort DNA through Gel Electrophoresis • Transfer DNA to a solid support • Methods of transferring • The Hybridization Reaction • Denatured and Nicked DNA • Radioactive Probe • Continuation of the Hybridization Reaction • Comparing DNA fingerprints January 28, 2003

  16. Cut the DNA

  17. Cut large genome into shorter DNA fragments with restriction enzymes. Enzymes will recognize four to six specific base sequences and cleave the DNA at these specific boundaries Cut DNA January 28, 2003

  18. Variable Number Tandem Repeats (VNTRs) • Variable Number Tandem Repeats-repeated sequences of base pairs found at the introns (the “useless” part of of the DNA strand). • VNTRs contain from 20-100 base pairs. An example of VNTRs January 28, 2003

  19. VNTRs cont. • Every human has unique VNTR sequence (because VNTRs are inherited genetically). • They may be used in the production of a DNA Fingerprint • The VNTRs must go through: Southern Blotting, probing, and a hybridization reaction in order to result in a DNA fingerprint. January 28, 2003

  20. Agenda Revisited • Collect the DNA • Isolate the DNA • Cut DNA • Variable Number Tandem Repeats (VNTRs) • Sort DNA through Gel Electrophoresis • Transfer DNA to a solid support • Methods of Transferring • The Hybridization Reaction • Denatured and Nicked DNA • Radioactive Probe • Continuation of the Hybridization Reaction • Comparing DNA fingerprints January 28, 2003

  21. Sort the DNA

  22. Sort DNA Through Gel Electrophoresis • Gel Electrophoresis separates DNA molecules by size NOT by molecular weight. • Prior to process, must first: • Prepare slab of gel material cast • Set gel up for electrophoresis by having electrodes apply an electric field. • DNA is slightly negative (REMEMBER!!!) Slab of agarose January 28, 2003

  23. Sorting DNA Through Gel Electrophoresis (Cont’d) • The DNA molecules will then be separated by size • In the gel agarose: • Negative (-) electrode is on left side, positive (+) electrode on right side • Since DNA molecules have a (-) charge (you already memorized that), they will want to move from left to right. January 28, 2003

  24. Gel has pores restraining larger molecules from moving all the way to the right side Hence, smaller DNA molecules will flow through quickly, this separates the molecules by SIZE Sorting DNA Through Gel Electrophoresis (Cont’d) DNA molecules moving through agarose. January 28, 2003

  25. Agenda Revisited • Collect the DNA • Isolate the DNA • Cut DNA • Variable Number Tandem Repeats (VNTRs) • Sort DNA through Gel Electrophoresis • Transfer DNA to a solid support • Methods of Transferring • The Hybridization Reaction • Denatured and Nicked DNA • Radioactive Probe • Continuation of the Hybridization Reaction • Comparing DNA fingerprints January 28, 2003

  26. Transfer the DNA

  27. Transferring DNA Onto a Solid Support • DNA is sorted into single strands either by heating or chemical treatment in gel. • After DNA molecules are separated by size, the protein must be transferred onto some solid support in preparation for hybridization. This process is called blotting. January 28, 2003

  28. Method of Transferring • DNA must be transferred onto a SOLID support. • A commonly used solid support is nitrocellulose paper (filter paper). January 28, 2003

  29. Electrophoresis – Capillary Blotting • The transferring process usually goes via electrophoresis or capillary blotting • Electrophoresis is the transfer separation of molecules by size • Capillary blotting is the process in which the molecules are transferred in a flow of buffer from wet filter paper to dry filter paper. Equipment used in Gel electrophoresis January 28, 2003

  30. Agenda Revisited • Collect the DNA • Isolate the DNA • Cut DNA • Variable Number Tandem Repeats (VNTRs) • Sort DNA through Gel Electrophoresis • Transfer DNA to a solid support • Methods of transferring • The Hybridization Reaction • Denatured and Nicked DNA • Radioactive Probe • Continuation of the Hybridization Reaction • Comparing DNA fingerprints January 28, 2003

  31. The Hybridization Reaction

  32. The Hybridization Reaction • Hybridization reaction-the binding of two genetic sequences, specifically the denatured and Nicked DNA and the radioactive probe. • Binding occurs between A and T and C and G through Hydrogen bonds. There are two hydrogen bonds between A and T and three H-Bonds between C and G. HOW am I supposed to Explain this thing??? January 28, 2003

  33. Denatured and Nicked DNA • However first DNA must be denatured. To denature DNA, the existing H-Bonds must first be broken through chemical processes or heating. This leaves a single strand of DNA whose bases are available for hydrogen bonding • Nicked DNA-DNA that has been cut in certain areas for further use. January 28, 2003

  34. Radioactive Probe Creation • How a radioactive probe is created • The nicked DNA strand is essentially repaired by the DNA polymerase, and at the same time, making it radioactive by including the C* bases. • The nicked DNA is then heated and split apart resulting in single stranded radioactive and non-radioactive pieces. The radioactive DNA piece is called the probe. Probe January 28, 2003

  35. The Hybridization Rxn continued • The single stranded radioactive probe can be used to see if the denatured DNA contains a sequence similar to that on the probe. January 28, 2003

  36. Hybridization Rxn cont. • If a positive match does comes up and the DNA probe contains a sequence similar to that of the denatured DNA, the two will form H-Bonds and bind. • Although if the fit between the two sequences is poor, there will be fewer H-Bonds. • The ability for low-homology probes to still bind to DNA sequences may be altered through varying amounts of saline solution or varying temperatures. January 28, 2003

  37. Hybridization Rxn cont. • Obtain some DNA polymerase, place radiolabeled DNA into a tube • Make horizontal breaks along a strand of DNA to be radiolabeled. While doing this, add individual nucleotides to the nicked DNA. January 28, 2003

  38. Hybridization Rxn cont. • Add DNA polymerase into tube (which now contains nicked DNA ready to be radiolabeled). • Once DNA polymerase is added, it will immediately be attracted to the nicks in the DNA and attempt to “repair” the DNA. In doing so, it will destroy all existing bonds in front of it and will place the new nucleotides (added earlier) behind it. • Every G base will bond with a C* base. January 28, 2003

  39. Hybridization Rxn cont. • Locate a specific VNTR sequence on a single stranded DNA fragment • Make a DNA probe out of DNA sequence • Labeling probe with radioactive compound • Letting probe bind to like DNA sequences on membrane • Use radioactive tag to find where probe has attached January 28, 2003

  40. Agenda Revisited • Collect the DNA • Isolate the DNA • Cut DNA • Variable Number Tandem Repeats (VNTRs) • Sort DNA through Gel Electrophoresis • Transfer DNA to a solid support • Methods of Transferring • The Hybridization Reaction • Denatured and Nicked DNA • Radioactive Probe • Continuation of the Hybridization Reaction • Comparing DNA fingerprints January 28, 2003

  41. Compare DNA Profile

  42. Visualize Banding by Exposure X-ray Film • Take a picture of probe stuck to its target on the membrane using specialized X-ray film • Place membrane on the special sheet of film for a short period of time • And you have a picture! January 28, 2003

  43. Thank You • Thank you for listening to my presentation! • I hope you now have clear understanding as to how to make a DNA profile! January 28, 2003

  44. Bibliography • http://merriam-webster.com/cgi-bin/dictionary • http://www.howstuffworks.com/dna-evidence.htm • http://www.botany.uwc.ac.za/mirrors/MIT-bio/bio/rdna/rdnadir.html • http://www.biology.washington.edu/fingerprint/dnaintro.html • www.accessexcellence.org/AB/GG/restriction.html • www-hhmi.princeton.edu/grp2/size of dna molecule.htm • www.frontiernet.net/~plasmid/pictures/ansvr.jpg • www.biology.washington.edu/ fingerprint/radio01.gif • esg-www.mit.edu:8001/esgbio/ rdna/probe.gif • www.hsa.gov.sg/hsa/Images/ cfs/dna_profiling2.jpg • www.labcorp.com/paternity/ body_index.html • criminaljustice.state.ny.us/ ops/history/bert_1.jpg • www.sun.ac.za/kie/unistel/medical_labs/ paternity3.htm • www.majintl.com/dogtag.htm January 28, 2003