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A C G C A C TT C A G AA C G C G T A C T G A C T G AA

2. 3. 4. A C G C A C TT C A G AA C G C G T A C T G A C T G AA. T G C G T G AA G T C TT G C G C A T G A C T G A C TT. Homework: Tuesday : Lab Notebook – with Serial Dilution & Spectrophotometer Wednesday : News article – specific and detailed for credit . Agenda: 4/28.

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A C G C A C TT C A G AA C G C G T A C T G A C T G AA

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  1. 2 3 4 ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT

  2. Homework: Tuesday: Lab Notebook – with Serial Dilution & Spectrophotometer Wednesday: News article – specific and detailed for credit Agenda: 4/28 Objective: To determine how DNA is used in forensic cases Human Genome – how people differ DNA Uses and Sources DNA Fingerprinting – Steps needed - Restriction enzymes - Gel electrophoresis - Polymerase Chain Reaction

  3. Class notebook

  4. DNA fingerprinting DNA forensics What we need to know: RFLP Restriction Fragment Length Polymorphism Restriction enzymes How/why does gel electrophoresis works? How/why does PCR work? Use of informatics/statistics • Digesting a DNA sample using restriction enzymes • Gel electrophoresis • Process: running gels • Data analysis • Polymerase Chain Reaction • Process • Interpretation • Solving the Case • Paternity Case - Blacketts

  5. The Human Genome The sequence of bases make up our genes. The Human Genome Project determined the order of each of these bases in all of our genes. Also found that most DNA is not coding for genes. There are many areas in which bases are repeated.

  6. Facts & Figures about DNA How many bases are there in the human genome? • 3,000 • 300,000 • 3 million • 3 billion • 3 trillion

  7. Facts & Figures about DNA How many bases are there in the human genome?

  8. Facts & Figures about DNA How many bases are there in the human genome? • 3,000 • 300,000 • 3 million • 3 billion • 3 trillion

  9. Facts & Figures about DNA How many bases are there in the human genome? 3,000,000,000

  10. Facts & Figures about DNA We are not all exactly the same – What percent of your DNA is similar to any other person in the world?

  11. Facts & Figures about DNA We are not all exactly the same – What percent of your DNA is similar to any other person in the world? • 99.9% • 98% • 90% • 60% • 10%

  12. Facts & Figures about DNA We are not all exactly the same – What percent of your DNA is similar to any other person in the world? • 99.9% • 98% • 90% • 60% • 10%

  13. Facts & Figures about DNA We are not all exactly the same – What percent of your DNA is similar to any other person in the world? 3 MILLION bases are different!

  14. Facts & Figures about DNA • Forensic scientists focus on these variable regions to generate a “DNA fingerprint” for each individual

  15. Summary – Nuclear DNA

  16. DNA Use in Forensic Cases • Most are rape cases (>2 out of 3) • Looking for match between evidence and suspect • Must compare victim’s DNA profile Challenges • Mixtures must be resolved • DNA is often degraded • Inhibitors to PCR are often present

  17. Human Identity Testing • Forensic cases -- matching suspect with evidence • Paternity testing -- identifying father • Historical investigations • Missing persons investigations • Mass disasters -- putting pieces back together • Military DNA “dog tag” • Convicted felon DNA databases

  18. YouTube – DNA forensics – 4 videos • https://www.youtube.com/watch?v=dXYztbkMXwU&list=PLC0B027FC81C82602 – 2 minute overview • Includes CODIS – Story • https://www.youtube.com/watch?v=VF5s1loHxx4&list=PLC0B027FC81C82602 • https://www.youtube.com/watch?v=8w_VJ4G7qiw&list=PLC0B027FC81C82602 • https://www.youtube.com/watch?v=yWKbQH2P6og&list=PLC0B027FC81C82602

  19. What are some sources of DNA?

  20. Sources of Biological Evidence • Blood • Semen • Saliva • Urine • Hair • Teeth • Bone • Tissue • Mucus • Ear Wax

  21. DNA fingerprinting DNA forensics What we need to know: RFLP Restriction Fragment Length Polymorphism Restriction enzymes How/why does gel electrophoresis works? How/why does PCR work? Use of informatics/statistics • Digesting a DNA sample using restriction enzymes • Gel electrophoresis • Process: running gels • Data analysis • Polymerase Chain Reaction • Process • Interpretation • Solving the Case • Paternity Case - Blacketts

  22. Cutting the DNA with Restriction Enzymes

  23. Restriction Enzyme Digest DNA can be cut into smaller pieces by restriction enzymes that recognize very specific sequences of DNA. AGCTAGAATTCTTTACGCTCGGATGAATTCCACCTATCTCC

  24. Restriction Enzyme Digest DNA can be cut into smaller pieces by restriction enzymes that recognize very specific sequences of DNA. AGCTAGAATTCTTTACGCTCGGATGAATTCCACCTATCTCC AATTCTTTACGCTCGGATG AGCTAG AATTCCACCTATCTCC

  25. Multiple Restriction Enzymes Exist for Cutting DNA EcoRI GAATTC G AATTC PstI CTGCAG CTGCA G SmaI CCCGGG CCC GGG HindIII AAGCTT A AGCTT BamI GGATCC G GATCC HaeIII GGCC GG CC

  26. Separating the DNA fragments RFLP analysis

  27. Visualizing the DNA Restriction Fragments 1 – Ladder to determine size (number of base pairs in each segment) 2-7 samples from suspects or victims

  28. DNA Restriction Enzymes • Evolved by bacteria to protect against viral DNA infection • Endonucleases = cleave within DNA strands • Over 3,000 known enzymes

  29. Enzyme Site Recognition Restriction site Palindrome • Each enzyme digests (cuts) DNA at a specific sequence = restriction site • Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC) Fragment 2 Fragment 1

  30. 5 vs 3 Prime Overhang Enzyme cuts • Generates 5 prime overhang

  31. Common Restriction Enzymes EcoRI – Eschericha coli – 5 prime overhang Pstl – Providencia stuartii – 3 prime overhang

  32. The DNA DigestionReaction Restriction Buffer provides optimal conditions •NaClprovides the correct ionic strength • Tris-HCI provides the proper pH • Mg2+ is an enzyme co-factor

  33. DNA DigestionTemperature Why incubate at 37°C? • Body temperature is optimal for these and most other enzymes What happens if the temperature is too hot or cool? • Too hot = enzyme may be denatured (killed) • Too cool = enzyme activity lowered, requiring longer digestion time

  34. Restriction Fragment Length PolymorphismRFLP PstI EcoRI GAATTC GTTAAC CTGCAG GAGCTC Allele 1 1 2 3 CGGCAG GCGCTC GAATTC GTTAAC Allele 2 3 Fragment 1+2 Different Base Pairs No restriction site M A-1 A-2 Electrophoresis of restriction fragments M: Marker A-1: Allele 1 Fragments A-2: Allele 2 Fragments +

  35. AgaroseElectrophoresisLoading • Electrical current carries negatively-charged DNA through gel towards positive (red) electrode Buffer Dyes Agarose gel Power Supply

  36. AgaroseElectrophoresisRunning • Agarose gel sieves DNA fragments according to size – Small fragments move farther than large fragments Gel running Power Supply

  37. Analysis of Stained Gel Determine restriction fragment sizes • Create standard curve using DNA marker • Measure distance traveled by restriction fragments • Determine size of DNA fragments Identify the related samples

  38. Molecular Weight Determination Fingerprinting Standard Curve: Semi-log Size (bp) Distance (mm) 23,000 11.0 9,400 13.0 6,500 15.0 4,400 18.0 2,300 23.0 2,000 24.0

  39. Polymerase Chain Reaction (PCR) ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT PCR can make many copies in a very short period of time

  40. Polymerase Chain Reaction (PCR) ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT Heat to 94°C: Denature Strands of DNA

  41. Polymerase Chain Reaction (PCR) ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAA TGCGTGAAGTCTTGCGCATGACTGACTT TGACTGAA Cool to 55°C: Allow primers to anneal

  42. Polymerase Chain Reaction (PCR) ACGCACTTCAGAACGCGTACTGACTGAA ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT TGCGTGAAGTCTTGCGCATGACTGACTT Heat to 72°C: New DNA strand is synthesized

  43. Polymerase Chain Reaction (PCR) ACGCACTTCAGAACGCGTACTGACTGAA ACGCACTTCAGAACGCGTACTGACTGAA ACGCACTTCAGAACGCGTACTGACTGAA ACGCACTTCAGAACGCGTACTGACTGAA ACGCACTTCAGAACGCGTACTGACTGAA ACGCACTTCAGAACGCGTACTGACTGAA ACGCACTTCAGAACGCGTACTGACTGAA ACGCACTTCAGAACGCGTACTGACTGAA ACGCACTTCAGAACGCGTACTGACTGAA ACGCACTTCAGAACGCGTACTGACTGAA ACGCACTTCAGAACGCGTACTGACTGAA ACGCACTTCAGAACGCGTACTGACTGAA ACGCACTTCAGAACGCGTACTGACTGAA TGCGTGAAGTCTTGCGCATGACTGACTT TGCGTGAAGTCTTGCGCATGACTGACTT TGCGTGAAGTCTTGCGCATGACTGACTT TGCGTGAAGTCTTGCGCATGACTGACTT TGCGTGAAGTCTTGCGCATGACTGACTT TGCGTGAAGTCTTGCGCATGACTGACTT TGCGTGAAGTCTTGCGCATGACTGACTT TGCGTGAAGTCTTGCGCATGACTGACTT TGCGTGAAGTCTTGCGCATGACTGACTT TGCGTGAAGTCTTGCGCATGACTGACTT TGCGTGAAGTCTTGCGCATGACTGACTT TGCGTGAAGTCTTGCGCATGACTGACTT TGCGTGAAGTCTTGCGCATGACTGACTT PCR can make many copies in a very short period of time

  44. How do we generate a DNA fingerprint? …After amplification of the variable regions through PCR

  45. FBI’s CODIS DNA Database Combined DNA Index System • Used for linking serial crimes and unsolved cases with repeat offenders • Launched October 1998 • Links all 50 states • Requires >4 RFLP markers and/or 13 core STR markers • Current backlog of >600,000 samples

  46. 13 CODIS Core STR Loci with Chromosomal Positions TPOX D3S1358 TH01 D8S1179 D5S818 VWA FGA D7S820 CSF1PO AMEL D13S317 AMEL D16S539 D18S51 D21S11

  47. Overview • Basic – DNA Fingerprinting • Overview: 6 min. Bozeman Science

  48. Use of Short Tandem Repeats • Non-coding sections (do not code from proteins) • Inherited from parents • Individuals have 2 copies (alleles)

  49. 13 CODIS Core STR Loci with Chromosomal Positions TPOX D3S1358 TH01 D8S1179 D5S818 VWA FGA D7S820 CSF1PO AMEL D13S317 AMEL D16S539 D18S51 D21S11

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