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DNA Typing - PCR

DNA Typing - PCR. Invented in 1987. PCR offers the possibility of increased sensitivity. It requires 50 times less sample than RFLP analysis. DNA sample, DNA polymerases, nucleotides, and a primer are placed in a DNA Thermal Cycler. Discriminates to 1 in 5000. DNA Typing - PCR.

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DNA Typing - PCR

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  1. DNA Typing - PCR Invented in 1987. PCR offers the possibility of increased sensitivity. It requires 50 times less sample than RFLP analysis. DNA sample, DNA polymerases, nucleotides, and a primer are placed in a DNA Thermal Cycler. Discriminates to 1 in 5000.

  2. DNA Typing - PCR Primers recognize small pieces of DNA that are complimentary to specific sequences of bases in the DNA sample.

  3. DNA Typing - PCR HLA DG alpha system (now called DQA1) contains 28 different primers. Polymarker (PM) has five additional genetic markers.

  4. DNA Typing – PCR analysis

  5. PCR

  6. DNA Typing - STR STR – Short Tandem Repeats The newest method of DNA typing. The FBI started using it in 1998. Still in use today. In combination with PCR (DQA1 and Polymarker) it is more discriminating (1 in 25,000,000) and less time consuming than RFLP. It also requires smaller sample sizes than RFLP.

  7. DNA Typing - STR STRs are locations (loci) on the chromosome that contain short sequence elements that repeat themselves within the DNA molecule.

  8. DNA Typing - STR The repeating sequence typically contains 3 to 7 bases. The sequence can repeat 3 to 65 times. HUMTH01 is a commonly used STR. It contains the A-A-T-G sequence. Seven variants (5 –11 repeats) have been identified in the human genome.

  9. DNA Typing - STR STRs can be amplified using PCR. There are hundreds of different types of STRs that can be used for DNA typing. Using more than one STR at a time is called multiplexing.

  10. Y-STRs • A set of specific STR markers associated with only the male Y chromosome has recently been developed • Y-STR typing can unambiguously determine what a male has contributed to a mixed sample • Often used in rape investigations because it can distinguish male DNA from female DNA

  11. Mitochondrial DNA

  12. Mitochondrial DNA Nuclear DNA – 1 per cell Mitochondria – 100 – 1000 per cell mtDNA is inherited solely from your mother. Therefore, others in your family have the same mtDNA.

  13. Mitochondrial DNA mtDNA has a loop configuration. It contains the information for 37 proteins. Two regions are highly variant: Hypervariable Region 1 (HV1) and Hypervariable Region 2 (HV2).

  14. Mitochondrial DNA The analytical technique of analysis of mtDNA, (sequencing), is very tedious, costly, and time consuming. Therefore, only a few labs are equipped to analyze mtDNA.

  15. Mitochondrial DNA Why do it then? mtDNA can be obtained from hair, bones, and very small tissue samples. mtDNA has been used to identify victims of mass destruction and unknown war victims.

  16. The Combined DNA Index System CODIS – A national computerized database containing the results of DNA analysis of sex offenders. The federal DNA Identification Act of 1994 initiated the nation wide project with $40 million in funding.

  17. The Combined DNA Index System Currently, thirteen STRs are entered in to CODIS for each DNA entry. The data base has been very effective in aiding investigators solve many crimes.

  18. Abandoned DNA • Police can offer suspect a drink or cigarette, then later collect the DNA from the item without the suspect’s knowledge • The Fourth Amendment protects against “unreasonable search” • However, courts have ruled that DNA testing on abandoned items does not violate an individual’s reasonable expectation of privacy

  19. Exonerations • As of May 2008, 216 post-conviction DNA exonerations have been made in the U.S. • The true perpetrators have been identified in 82 of the DNA exoneration cases • Preservation of evidence by state police is crucial to a DNA exoneration

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