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Gel Electrophoresis

Gel Electrophoresis. A molecular biology tool. Purpose . To separate and analyze/compare fragments of DNA. 4 Main Steps:. 1) Restriction Enzymes: Either one or several R.E.’s is added to a sample of DNA. Enzymes cut them into fragments. 2) Prepare Gel:

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Gel Electrophoresis

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  1. Gel Electrophoresis A molecular biology tool

  2. Purpose • To separate and analyze/compare fragments of DNA

  3. 4 Main Steps: • 1) Restriction Enzymes: • Either one or several R.E.’s is added to a sample of DNA. • Enzymes cut them into fragments

  4. 2) Prepare Gel: • A gel with consistency similar to gelatin (or pGreen agar) is formed • Small comb placed halfway into one end of gel to create DNA “wells” • Comb wiggled out leaving vacant wells

  5. 3) The Electric Field: • Gel placed in electric field where one end of gel is + and one –

  6. 4) The Fragments Move: • DNA has negatively charged phosphates which will move towards + end of plate • Gel provides some resistance • ** Smaller fragments move further! • (like snakes moving thru netted swimming pool)

  7. After gels run… • Gel is stained with dye (ethidium bromide – carcinogenic OR methyl blue) • Will see many bands • Bands depend on starting DNA and r.e. used

  8. Loading Dye • Blue colored solution used when loading DNA so you know when to shut off electric current • Usually size marker run along with loading dye and DNA fragments

  9. Possible analysis 2 • Can compare DNA samples run in SAME gel

  10. DNA Fingerprints aka DNA Profiles

  11. Definition • A pattern of bands made up of specific fragments from an individual’s DNA

  12. How are they used? • To establish whether 2 individuals are related • To determine how closely related 2 different species are • To help solve crimes: • Match crime scene DNA w/ suspect DNA

  13. How to Make a DNA Fingerprint~ PCR • Requires 20 ng DNA (can be somewhat degraded) • Denaturization by heat • hybridization (complementary pairing of short primers) http://www.accessexcellence.org/AB/GG/nucleic.html 3. DNA synthesis (by DNA polymerase) • Process repeats • http://www.accessexcellence.org/AB/GG/polymerase.html

  14. How to Make a DNA fingerprint • 1) RFLP (restriction fragment length polymorphism) analysis • Involves extracting DNA from a specimen of blood or other tissue • Cutting it into fragments using r.e.’s • Number and length of fragments vary from person to person

  15. What is a Polymorphism? • Variations in DNA sequence between individuals • sequences with the highest degree of polymorphism are very useful for DNA analysis in forensics cases and paternity testing

  16. RFLP’s (1st generation tech.) • Phased out in 1999 (started in ’85) • Looked at 5 areas of DNA (non-coding/junk areas) • Required 400 ng non-degraded DNA • Time consuming, labor intensive • Took several weeks, months for results • Use radioactive labels & X ray film – “Southern Blotting”

  17. RFLP Allele Analysis

  18. How to Make DNA fingerprint • 2) Short tandem repeats • Used presently • Few base prs. (10-100) repeated a # of times • Requires 2.5 ng or less • Can use badly degraded DNA (remains) • Extremely sensitive • Automated technique ~ 2 days to a week • Highly specific profiles unique to the individual

  19. STR’s • Target site identified by primers binding to the correct locus • 13 core CODIS "Short Tandem Repeat" loci used for the national DNA databank • CODIS = Combined DNA Index System • PCR produces millions of copies of STR locus • STR fragments separated by electrophoresis and analyzed by computer (1/2 hour)

  20. Probability of DNA Profiles • The probability (P) for a DNA profile is the product of the probability (P1, P2, ... Pn) for each individual locus, i.e. • Profile Probability = (P1) (P2) ... (Pn) • The probability can be an extremely low numbers when all 13 CODIS STR markers are included in the DNA profile!

  21. Frequency Calculation Example • Blackett Activity • Bob Blackett calculated his own profile probability at 1.3 times 10-16, or no more frequent than 1 in 7.7 quadrillion individuals (7.7 million billion), which is more than a million times the population of the planet.

  22. How do RFLP’s and STR’s differ? • Restriction fragments containing STR’s vary in size among individuals b/c of differences in STR lengths, rather than b/c of diff. # of restriction sites within that region of the genome as in RFLP’s

  23. Review • Review electrophoresis simulation • Forensic DNA analysis

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