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DNA Fingerprinting

DNA Fingerprinting. Use of DNA to Determine Identity. DNA controls production of proteins Results in phenotype (eye color, facial features) Contributes to structure and function DNA – 3 billion base pairs No other person on planet has same code (except identical twins!)

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DNA Fingerprinting

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  1. DNA Fingerprinting

  2. Use of DNA to Determine Identity • DNA controls production of proteins • Results in phenotype (eye color, facial features) • Contributes to structure and function • DNA – 3 billion base pairs • No other person on planet has same code (except identical twins!) • Only 0.1% of DNA differs from person to person • But the regions that do vary provide a true genetic blueprint

  3. Use of DNA to Determine Identity • DNA obtained from skeletal remains • DNA purified • If degraded – amplified w/ PCR • RFLPs compared to determine final identity of missing person

  4. What are some sources of DNA Evidence? • Skin cells • Hair • Blood • Semen • Anything with DNA

  5. DNA Fingerprinting • AKA - DNA profiling analysis or DNA typing • Electrophoretic analysis of DNA fragment sizes generated by restriction enzymes • Provides accurate, unambiguous identification of source DNA samples

  6. Restriction Enzymes • Endonucleases that cut phosphate bonds • Break bonds between deoxyribose and phosphate • Attach to DNA and “read” nucleotides • Cut at specific sequences • Over 3000 types

  7. Restriction Enzymes • Named after the organisms from which they were discovered / isolated • Eco RI – Escherichia coli RY13 • Hind III – Haemophilus influenzae R4 • Bam HI – Bacillus amyloliquefaciens H

  8. Eco RI Function Eco RI 5` - G A A T T C – 3` 3` - C T T A A G – 5` 5`- G A A T T C – 3` 3` - C T T A A G – 5` DNA fragments with “sticky” ends

  9. Restriction Enzymes • Forensic labs – use at least 2 restriction enzymes • 4-base and 5-base (sometimes others) • Size of DNA fragments – depends on distance between recognition sites • Longer DNA molecule – the greater probability that a specific recognition site will occur

  10. Restriction Enzymes • Average human chromosome = 100 million bp • Eco RI – 6 bp recognition site • Probability – 1 site / 4096 bp • Cut human DNA into ~25,000 fragments

  11. Restriction Enzymes • No two individuals have same pattern of restriction enzyme recognition sites • Each person – unique genotype (different alleles) • Mutations / Insertions / Deletions • Changes distribution and frequency of restriction enzyme recognition sites

  12. After Restriction Digest • Analyze DNA fragments on agarose gel • DNA fragments separated by molecular weight (size) due to net negative charge on phosphates • Restriction enzyme cleavage of relatively small DNA molecules – key to RFLP analysis • If DNA fragments are too large – can’t see RFLP pattern

  13. Sample RFLP Patterns Suspect RFLP #4 Control RFLP Suspect RFLP #1 Suspect RFLP #3 Suspect RFLP #5 Suspect RFLP #6 Molecular Marker Suspect RFLP #2

  14. Restriction Enzyme Digestion • Be sure to label all reaction tubes (4) • 10 μl of enzyme reaction buffer • 15 μl of DNA sample from missing person • 15 μl of Restriction Enzyme • Incubate at 37°C for ~60 minutes • Add 5 μl of Gel Loading Solution after incubation

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