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DNA typing

DNA typing. Restriction Fragment Length Polymorphism (RFLP). An older method for DNA analysis DNA is cut by restriction enzymes -enzymes made by bacteria. (ex. EcoRI and Pstl ). These enzymes cut the DNA at specific sites.

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DNA typing

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  1. DNA typing

  2. Restriction Fragment Length Polymorphism (RFLP) An older method for DNA analysis DNA is cut by restriction enzymes -enzymes made by bacteria. (ex. EcoRI and Pstl). These enzymes cut the DNA at specific sites. These pieces or fragments of DNA are then separated by size through a process called gel electrophoresis- the smaller fragments will move faster through the gel.

  3. Radioactively Labeled Probes: In order to visualize these separated fragments they are transferred to a nylon sheet and treated with a radioactively labeled probe containing a complementary base sequence to the RFLP being identified For example: to recognize a TAG sequence a probe with an ATC sequence would be used because it is complementary and will bind to this site.

  4. The DNA RFLP typing process

  5. Sir Edwin Southern Southern Blot Technique: http://www.bio.davidson.edu/courses/genomics/method/Southernblot.html

  6. RFLP If one probe is used the DNA pattern will show two bands one from each chromosome. When comparing DNA fragment patterns of two or more specimens, one looks for a match between band sets. More than one probe is typically used to increase the degree of discrimination (ones that recognize different repeating DNA segments)

  7. Probability and RFLP Analysis If four different probes are selected which each has a frequency of occurrence of one out of a hundred in the population than the combined frequency would be 1/100 x 1/100 x 1/100 x 1/100 = 1/100,000,000

  8. Case Example: RFLP was the first scientifically accepted protocol in the US used for the characterization of DNA Used in the Impeachment trial of Bill Clinton BUT New technology is better

  9. PCR Polymerase Chain Reaction A technique used to copy or multiply DNA strands Evidence is often in minute quantities so PCR is very useful RFLP strands are too long - PCR is best used with shorter strands.

  10. Kary Mullis

  11. Polymerase Chain Reaction (PCR)

  12. STR Analysis Dominant means of analysis is called SHORT TANDEM REPEAT (STR) analysis. 95% of the DNA is “junk” Short tandem repeats are sections of junk DNA with repeating sequences. Only a couple hundred base pairs versus RFLP’s which can have thousands

  13. Figure 9–11  Variants of the short tandem repeat TH01. The upper DNA strand contains six repeats of the sequence A–A–T–G; the lower DNA strand contains eight repeats of the sequence A–A–T–G.

  14. STR analysis: Benefits: shorter strands are more stable and can be amplified FBI has chosen 13 STR locations for use in STR analysis To find the probability of having multiple patterns of repeats the percentage of each pattern is multiplied together.

  15. Forensic STR Analysis

  16. Sex determination The sex of the DNA contributor can be determined by looking at the amelogenin gene located on both the X and the Y chromosome. The gene is for tooth pulp. It is shorter by six base pairs on the X than on the Y chromosome When amplified by PCR and separated by electrophoresis, males show two bands and females only show one.

  17. Y-STRs The Y chromosome is male specific. Paired with the X chromosome. More than 20 different Y-STR markers have been identified. Females have two X chromosomes so will not have any Y markers. Only one band for each STR type per person. Useful for sexual assault cases that involve multiple males.

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