Forensic DNA Analysis Criminalist Harry Klann, DNA Technical Leader DNA Detail Los Angeles Police Department Scientific Investigation Division PowerPoint presentation by Criminalist Carl Matthies
Objectives of Forensic DNA Testing s To link an individual to a crime scene/criminal act s To exonerate suspects s To identify victims of mass disasters
s A Brief Historys Contemporary Forensic DNA Testings Casework Applicationss Questions?
Early 1980s: Restriction FragmentLength Polymorphism (RFLP) s Genetic variation in the distance between restriction enzyme sites s Template DNA digested by enzymes, electrophoresed, detected via Southern blottingSir Alec Jeffreys s Power of discrimination in the range of 106-108 for a six probe analysis
Mid-1980s: The Colin Pitchfork Case s Two young women raped and murdered in Narborough, England s 5,000 local men are asked to provide blood/saliva samples s 1st exoneration and conviction on forensic DNA evidence
The Catch:s RFLP testing requires a relatively large amount of HMW DNA (~50ng = thousands of cells)s Not ideal for forensic evidence, in which small, degraded samples are common
PCR To The Rescue! s Polymerase Chain Reaction = molecular Xeroxing s Three temperature phases, carried out in a Thermal Cycler, replicate or “amplify” the desired DNADr. Kary MullisEccentric Genius fragment(s)
PCR (cont’d)s First forensic application is the DQα locus, later multi-plexed with Polymarker™ loci using dot-blot detection methods Works with lower quantity (1-2ng), lower quality sampless Power of discrimination goes from 102-106...not good enough for databasing
The Current Method of Choice:Autosomal Short Tandem Repeats s Non-coding, tetranucleotide sequences which vary greatly from person to person in the number of repeating units s Requires <1ng of DNA to type 13-15 STR loci s Power of discrimination ranges from 1014-1023. World population is 109 so bring on the database!
Applied Biosystems 310 Genetic Analyzer
The Process In a NutshellAmplified DNA samples are injectedinto a capillary. Fluorescent tags onthe DNA fragments are excited by alaser as they pass a window in thecapillary, the fluorescence is recordedby a camera, and this signal isconverted into a “peak” by thecomputer software.
STR dataX, Y, XY
STR data (cont’d)
STR data (cont’d) STR TYPING SUMMARY SHEETDate: DNA Analyst / Serial #: DR #:9/24/1999 MATTHIES V9780 00-00-00001 Item # AMEL D3S1358 vWA FGA D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820 D16S539 THO1 TPOX X, Y 17 8, 10 25(S) 15, 17 23, 26 14, 15 26 12, 15 10 9, 13 9, 10 8, 9 9, 10 X, Y 17 8, 10 X 15, 17 28, 11 25(E) 16, 18 19, 26 15 14, 16 8, 13 12 11, 12 7, 8 11 X 15, 17 32.2 11 X 15, 17 28, 11VICTIM 16, 18 19, 26 15 14, 16 8, 13 12 11, 12 7, 8 11 X 15, 17 32.2 11 X, Y 17 8, 10SUSPECT 15, 17 23, 26 14, 15 26 12, 15 10 9, 13 9, 10 8, 9 9, 10 X, Y 17 8, 10 “The DNA profile obtained from Item 25(S) matches the DNA profile of the suspect. The combination of genetic marker types exhibited by Item 25(S) and the suspect occurs in approximately one in one hundred quadrillion (1017) individuals…”
How are these astronomical figures derived?The product rule: combined probability of a series of independentevents is determined by multiplying the probabilities of each event.STR loci are inherited independently (unlinked)Homozygous loci: p2 (same allele inherited from mother and father)Heterozygous loci: 2pq (either allele could be inherited from eitherparent)p(17)2 x 2p(15)q(17) x 2p(23)q(26)….(.223)2 x 2(.083)(.25) x 2(.14)(.02) = .000013, which is equivalentto a probability of one in 76,000 using just 3 of the 13 loci!
STR Artifacts-A (“minus A”): Incomplete addition ofnucleotide ‘A’ by DNA polymerase;results in a peak that is one base pairsmaller than allele peak.
STR ArtifactsStutter: Slippage of DNA polymerase;results in a peak that is four base pairs(one repeat unit) smaller than allele peak.
STR ArtifactsPull-up: Incomplete filtration of spectraloverlap in fluorescent detection system.
DNA MixturesWhen more than one source of DNA is detected in a sample,assignment of genotypes becomes more difficult.
Degraded/Trace DNA SamplesLarger alleles “drop-out” when template DNA is low inquantity or quality, reducing certainty of genotypes.
The Combined DNA Index System (CoDIS) s A database of DNA profiles from violent felons and crime scene samples s Laws concerning who is eligible for the database vary from state to state s Database currently contains about 2,038,470 felons and 93,956 crime scene profiles (19,00 hits so far)
The Mystical Power of CoDIS s Extremely powerful investigative tool, linking crimes, and pulling suspects out of thin air! s Can prevent, as well as solve crimes!
The Dark Side of CoDIS (What the FBI doesn’t want you to know.) s DNA mixtures and degraded DNA profiles have lead to spurious matches s Stringent laws explicitly permit databasing innocent people s Adding arrestees to database violates presumption of innocence s However, the prosecution rate on case to offender matches is shockingly low! (~10%)
LAPD CoDIS Statistics s 177/142 Case-to-Offender matches s 100 Case-to-Case hits with 42 as yet unidentified suspects s 28 DA “rejects” s 9 Convictions; charges filed in 28 more; 4 defendants plead guilty s 280 investigations aided…
“Specialized” PCR-based systems s mtDNA s Y-STRs s SNPs
Mitochondrial DNA (mtDNA)
Mitochondrial DNA (mtDNA) Pross Single-cell sensitivity because each cell contains ~1000 mitochondrias Especially useful for shed hairs, burnt remainss Can be used to establish kinship directly because entire complement of mtDNA is maternally inherited
Mitochondrial DNA (mtDNA) Conss Single-cell sensitivity because each cell contains ~1000 mitochondria = very high contamination risk!s Heteroplasmy - more than one mtDNA type manifesting in different tissues in the same individuals Lower power of discrimination - maternal relatives all share the same mtDNA
Y-STRs Problem:s ~99% of violent crimes are committed by mens DNA Mixtures of male suspect and female victim can pose an analytical challenge, especially when the female contribution is much greater than the male = preferential amplification
Y-STRs Solution:s Test for markers found only on the Y- chromosome. Only male DNA is amplified!
Y-STRs “Khan” Arguments Lower power of discrimination - paternal relatives all share the same Y-STR haplotype (“Wicked Uncle Ernie” Defense)s 10% of Central Asian males share the same Y-STR haplotype, thought to belong to Genghis Khan
Single Nucleotide Polymorphisms (SNPs) s Point mutations (base substitutions) found in 1% or more of the population s 1.8 million identified in human genome s Detected on micro-array plates with fluorescent tags (all or nothing response)
SNPs (cont’d)s ~50 SNPs provides same power of discrimination as 13 STR locis Certain SNPs used as predictors of ancestry/ethnicity by a private sector lab (DNA Witness)
Flawed logic in the use of SNPs for predicting ancestry Scenario 1: SNP profile inconsistent with subject’s reported ancestry ¿ subject’s family history is inaccurate ¿ results are indisputable Scenario 2: SNP profile inconsistent with subject’s physical appearance ¿ ad hoc human migration explanation ¿ results are indisputable
Other SNP Flawss Privacy issues - unlike STRs, SNPs can be correlated with susceptibility/resistance to diseasess Requires a relatively large quantity of DNA for robust assay