DNA 101. DNA 101:Kate Philpott and Jennifer Friedman

Transcription

1 DNA 101 DNA 101:Kate Philpott and Jennifer Friedman

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4 DNA found in cells of your body cell nucleus DNA Found in cells once they leave your body too

5 CONSIDER THE SOURCE Blood* Semen* Hair Bone Saliva* Urine* Tooth Skin/tissue * Before DNA testing, these & other bodily fluids can be classified based on type using serological testing Presumptive testing: a particular bodily fluid is possibly the source, but there are a number of substances that cause false positives Confirmatory testing: much more specific (false positives are still possible/less plausible)

6 DNA is sometimes stored as a CHROMOSOME Most human cells contain 46 chromosomes (23 pairs): 22 pairs of chromosomes 2 sex chromosomes (X,Y): XY in males. XX in females.

7 ~ 6pg* of DNA. 2 copies (46 total) ~ 3pg of DNA. Sex cells (egg/sperm) 1 copy (23 total) ~ 6pg of DNA. Combine to form child with 2 copies *1 picogram (pg) = gram (one trillionth of a gram) 1000pg = 1nanogram (ng) (one billionth of a gram) 7

8 8 What are those crazy words they re always saying? Locus location of an allele on a chromosome; current tests target 16 to 24 locations. Plural: loci. Locus 1 Allele a variant form occurring at location. Typically a person will have 2 alleles (either different or the same) at each locus, one on each of their 2 chromosomes, eg. 15 and 18. Locus 2 At this locus, you could have allele 1 or 2 or 3, etc.

9 13 CODIS Core STR Loci + 7 loci TPOX D3S1358 D5S818 FGA CSF1PO D8S1179 D7S820 TH01 VWA AMEL D13S317 D16S539 D18S51 D21S11 AMEL

10 Forensic DNA Testing- STRs Short Tandem Repeats places where short DNA sequences repeat repeat repeat count the number of repeats at a particular location (locus) on each of 2 chromosomes; those are your alleles at those loci number of repeats varies between people

11 Short Tandem Repeats What are you looking at? 4 6 DNA Profile = 4,6

12 More vocabulary Genotype a description of the alleles present in an individual at a particular locus. Ex: 4, 6 Homozygous the condition of having two alleles which are the same. Ex: 4, 4 Heterozygous the condition of having two alleles which are different. Ex: 4, 6

13 Locus A Locus Genotype A 4, 5 B 3, 6 Allele 4 Allele 5 Homologous pair of chromosomes 4 repeats 5 repeats Allele 3 Allele 6 Homologous pair of chromosomes 3 repeats Locus B 6 repeats

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17 So how do we go from this to this? Locus Genotype A 4, 5 B 3, 6

18 Sampling Extraction Quantitation Amplification Capillary electrophoresis (Magic Science Box) Data analysis

19 SAMPLING Swab of suspected blood from scene, cutting of stain from cloth, etc. Evidence submission form from case: The victim was located a few feet from the firearm when officers arrived on the scene. From the crime scene it would appear that suspects may have been shooting at each other. DNA would help eliminate the victim as a possible shooter. Evidence for submission: 1.1: Wet and dry swab of red stain on the slide and grip and also the magazine 1.2: Wet and dry swab of the trigger, grip, slide and magazine

20 Extraction of DNA Blood DNA Chem + Heat break open the cells, releasing DNA Spin broken cell bits to the bottom How do we get the DNA out?

21 Swab of suspected semen from CW or from scene, cutting of stain from clothing, etc.

22 Remove a portion of the mixed stain DIFFERENTIAL EXTRACTION (SPECIAL TYPE OF EXTRACTION) SDS, EDTA and proteinase K (cell lysis buffer) Incubate at 37 o C Centrifuge sperm mixed with CW s epithelial cells SDS, EDTA and proteinase K + DTT Incubate at 37 o C Centrifuge DTT lyses sperm heads sperm pellet REMOVE supernatant Sperm Fraction sperm pellet Epithelial Fraction Figure 3.2, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition 2005 Elsevier Academic Press

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24 Importance of DNA Quantitation - Goldilocks Effect DNA amount (log scale) 100 ng 10 ng -A +A Too much DNA 1 ng 1.0 ng STR Kits Work Best in This Range 0.5 ng Well-balanced STR multiplex 0.1 ng 0.01 ng Too little DNA

25 ng/ul x 50 ul = ng 1.5 ng/ul x 50 ul = 75 ng

26 Amplification (making copies) Primers DNA bases Taq polymerase DNA

27 Extracted DNA

28 Two copies of each locus, e.g. a 6 allele and a 10 allele 10 6

29 Xerox copies of only the targeted regions or loci

30 Usually rounds of xeroxing.

31 100 cells produces 2.7 billion copies of each allele

32 Amplification PCR:Polymerase Chain Reaction 28 Cycles 1 Cycle 2 Cycles DNA 3 Cycles 4 Cycles 5 Cycles

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34 Analysis of amplified DNA DNA Profile Amplified DNA

35 Close-up of ABI Prism 310 Sample Loading Area Electrode Capillary Sample Wells Autosampler Tray

36 The Jell-O Race Amplified STR DNA injected onto column Electric current applied DNA pulled towards the positive electrode DNA separated out by size: Large STRs travel slower Small STRs travel faster Detector Window Color and migration time of STR detected and recorded as it passes the detector

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38 Suspect Reference Case 1 evidence (+) control Victim Ref. Ladder Case 2 evidence Blank (control)

39 STR procedure: complete separation Evidence sampled References sampled Evidence extracted References extracted Quantification Quantification Amplification Amplification Capillary electrophoresis (ABI 3130) Capillary electrophoresis (3130) Computer Analysis (e-data) Computer Analysis (e-data)

40 STR procedure: evidence & refs. Evidence sampled References sampled Evidence extracted References extracted Quantification Amplification Sampling & extraction of evidence & reference samples SHOULD be separated in TIME and/or SPACE Capillary electrophoresis (3130) Computer Analysis (e-data)

41 CONTROLS Samples that are included in a test under the same conditions as regular samples to monitor the performance of the test Positive: to ensure the test is functioning correctly Negative: to ensure no contamination is being read as true results

42 Detector Window

43 Locus Genotype D8 13, 14 D21 29, 30.2 D7 10, 11 CSF 12 D3 14, 15 THO1 7, 8 D13 12 D16 10, 13 D2 19, 20 D19 14, 16 vwa 14 TPOX 11 D18 13, 15 Amel X, Y D5 12 FGA 10, 22

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46 Allele Table = report of genotype Locus Evidence Item #1 (Cig butt at crime scene) Evidence Item #2 (knife handle) Suspect Reference buccal swab (Jennifer Friedman) Victim Reference blood card (Kate Philpott) D8S ,14 (12), 14 12,14 14,16 D21S , 30. 2, 31.2, , 31.2 D7S , CSF1PO 11,12 11, 12 11,12 11,12 D3S , 17, TH01 9.3, , ,10 7,8 D13S317 9,12 11, --- 9,12 11 D16S539 11,13 11, ,13 11,12 D2S , ,24 20,24 D19S433 12,14 12, 14 12,14 12,14 vwa 19 17, (18), TPOX 9,10 (8), 9, 10 9,10 8 D18S51 12,15 (12), (13), 15 12,15 13,15 AMEL X X, Y X X, X D5S818 8,11 11, --- 8,11 11,12 FGA 19,24 20, 23, 24, ,24 21,23

47 Range of conclusions Cannot be excluded Peaks between the compared STR profiles have the same genotypes and no unexplainable differences exist between the samples. Statistical evaluation of the significance of the match is usually reported with the match report. Excluded The genotype comparison shows profile differences that can only be explained by the two samples originating from different sources. Inconclusive Due to limited or overly complex DNA data, no conclusions can be made. An inconclusive does not mean maybe your client is there, it s a close call. It means the analyst decided the data developed from the evidence was not of sufficient quality to make any comparisons, and never compared the evidence DNA profile to the reference DNA profiles.

48 Statistical estimates: the product rule x x 2 = 0.1

49 Statistical estimates: the product rule 1 in 10 x 1 in 18 x 1 in 32 = in 5,760 1 in 510 x 1 in 13 x 1 in in 729,300 1 in 29 x 1 in 17 x 1 in 10 1 in 20,709,786,240,000 1 in 20 trillion 1 in 4,930

50 Comparing (unambiguous) electropherograms DIFFERENT Evidence (Bloodstain) Suspect reference

51 Comparing (unambiguous) electropherograms SAME Evidence (Bloodstain) Victim reference

52 Mixtures are often ambiguous SLIDE WITH DIFFICULT MIXTURE

53 Steps in mixture interpretation Step #1 Step #2 Step #3 Identify the Presence of a Mixture Designate Allele Peaks Identify the Number of Potential Contributors Step #4 Step #5 Step #6 Estimate the Relative Ratio of the Individuals Contributing to the Mixture Consider All Possible Genotype Combinations Compare Reference Samples From J.M. Butler (2005) Forensic DNA Typing

54 Step #1: Identifying a mixture Three or more alleles at a locus Many labs require three or more alleles at two or more loci Some labs will refer to an extra allele Peak height imbalance between alleles In theory, 2 alleles at a locus coming from the same person (a heterozygote) should be about the same height Many labs use 60% or 70% guideline Should be consistent across loci

55 4 PEAKS MIXTURE X>Y

56 Is it a MIXTURE?? 7 allele < 60% of 9.3 >2 alleles; PH imbalance D3 TH01 D13 D16 D2 CLIENT: 14,19 7,7 11,12 10,12 20,22

57 Step #1: Identifying a mixture Three or more alleles at a locus Many labs require three or more alleles at two or more loci Some labs will refer to an extra allele Peak height imbalance between alleles In theory, 2 alleles at a locus coming from the same person (a heterozygote) should be about the same height Many labs use 60% or 70% guideline Should be consistent across loci Quant results?

58 #2 Designating allele peaks: major source of ambiguity ARTIFACTS of the DNA testing process that may mimic alleles: stutter, blobs, spikes, pull-up Reading between THRESHOLDS the PEAK

59 #3 Identifying number of contributors No more than 4 alleles per locus 2 contributors? PHRs don t work Actually FOUR contribs

60 #4 Estimate ratio of contributors Relies on step three (id ing # contributors) Best to do where there is no allelic overlap Cannot always be calculated w/ confidence Difficult when more than 2 contributors

61 #5 Consider all possible genotypes Relies on steps 2, 3 and 4 to decide what is possible Goes toward interpretation and downstream statistics Maj: 15,18 Min: 15, 15 15, 16 15, 18 16, 16 16, 18 18, 18