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EDVOTEK 225 DNA Fingerprinting

EDVOTEK 225 DNA Fingerprinting. Spring 2010 Terry Kotrla, MS, MT(ASCP)BB. DNA Fingerprinting. Also called DNA profile analysis DNA typing Involves electrophoretic anlaysis of DNA fragment sizes generated by restriction enzymes More accurate an unambiguous then blood typing.

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EDVOTEK 225 DNA Fingerprinting

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  1. EDVOTEK 225DNA Fingerprinting Spring 2010 Terry Kotrla, MS, MT(ASCP)BB

  2. DNA Fingerprinting • Also called • DNA profile analysis • DNA typing • Involves electrophoreticanlaysis of DNA fragment sizes generated by restriction enzymes • More accurate an unambiguous then blood typing

  3. Restriction Enzymes • Restriction enzymes are endonucleases that catalyze cleavage of phosphate bonds • Require Mg-2 for activity • Generate 5’ phosphae and 3’ hydroxyl group • Endonuclease claves at specific sequence of bases. • Produce by bacteria

  4. Restriction Enzymes • Names after organism discovered in. • First 3 letters of genus • Strain or substrain • Roman numeral to designate one of the enzymes produced by the same organism.

  5. Restriction Enzymes • Require a specific double stranded sequence of nuceotide bases to cut DNA • Recognition sites usually 4-8 base pairs within or neat site • Frequently symmetrical, both DNA strands have same base sequence when read 5’ to 3’, ie, palindrome. • In forensics 2 most commonly used are Hae III and Hinf I.

  6. RFLP • Restriction Fragment Length Polymorphisms • http://tinyurl.com/2evjhfu

  7. Restriction Enzymes • Size of fragments depends on distances between recognition sites. • Long DNA molecule will have higher probability recognition site will be present. • Restriction enzyme with 6 bp recognition site expected to cut human DNA into approximately 750,000 different fragments

  8. Fragments • No 2 individuals have same pattern of recognition sites. • Alleles result in alternative expressions of genetic traits, dominant or recessive • Two copies of gene at given locus, mom & dad • Alleles have differences in base sequences • Mutations and deletions occur which eliminate palindromic site (figure 2)

  9. Repeats • Repetitive base sequences occur • Constitute large fraction of mammalian genome • Have no known genetic function • 10-15% of DNA consists of repeated short sequences. • Vary between individuals • When flanked by recognition sites the length of repeat will determine size of fragment generated.

  10. Electrophoresis • Used to analyze DNA fragments • DNA has negative charge. • Gel is a sieve which separate DNA fragments according to size, charge and shape. • Only size of DNA affects mobility. • Cleavage of large complex human DNA generates fragments which may exceed resolving capacity of gel. • Cleaved DNA will appear as a smear, no bands.

  11. Southern Blot • Electrophoresis is done. • Denature DNA fragments in alakali solution. • Double stranded fragments converted to single stranded form. • Trnafer DNA fragments to nitrocellulose (blotting) • Blotted DNA hybridized with labeled oligonucleotide DNA probe.

  12. Southern Blot • Probe • DNA fragment which is complementary to sequences found on human chromosomes • Labeled with a “reporter” to detect target • Probe incubated with blotted membrane and will hybridize to complementary sequences on membrane. • Allows only specific DNA fragments to be detected.

  13. Forensics • DNA samples collected at crime scene extracted from • Skin • Blood • Semen • Hair • Perform RFLP analysis • If pattern matches it will prove beyond a reasonable doubt that suspect was at crime scene. • Forensics uses different sets of probes to hybridize to different repetitious sequences to satisfy statistical probability for positive identification.

  14. DNA and PCR • PCR very sensitive, requires very little DNA • Allows increased speed for analyzing • Requires • DNA polymerase Taq polymerase • Four dinucleotides • Primers - two synthetic oligonuceotides 15-30 bp in length which correspond to start and end of DNA to be amplified (target) • Buffer with Mg2+

  15. PCR • Three phases • Heat to 92-96C to denature DNA • Cool to 45-65 to allow primers to anneal to separated strands • Heat to 72C to all extension, nucleotides added to primers.

  16. DNA Fingerprinting • Significant in court cases such as: • Murder • Rape • Physical battery • Results in acquittal or conviction • Process is standardized

  17. Procedure • PCR has been done • Have crime scene DNA and 2 suspects • Will treat all 3 samples with 2 restriction enzymes. • After enzyme treatment will electrophoreseand stain. • Analyze gels to determine guilty suspect, one whose DNA matches crime scene DNA

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