DNA Expert Witness. Nuclear DNA 10/23/14. Adams County Bar Association. DNA found in the nucleus of the cell is called nuclear DNA.

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1 Adams County Bar Association DNA Expert Witness Jenifer Smith, PhD Forensic Science Program Eberly College of Science Adams County Bench Bar Conference 10/31/14 Nuclear DNA DNA found in the nucleus of the cell is called nuclear DNA 1

2 10/23/14 DNA Structure Deoxyribonucleic Acid- DNA is a double helix strand that is composed of Deoxyribose (sugar group)- rails of the ladder Phosphate rails of the ladder Nitrogenous bases- rungs of the ladder 2

3 Why test nuclear DNA from evidence???? It can be retrieved from evidentiary stains and tissues DNA tests are available Unique to you unless you have an identical twin Quan44es of Extracted DNA Table 3.2, p. 49, BT 3

4 DNA Profiling is a Comparison Compare DNA types in evidence with DNA types in from reference samples from suspects or victims Reference samples may be a blood sample Reference samples may be a saliva sample If the types match, the source of the reference sample is a possible source of the DNA in the evidence If the types do not match, the source of the reference sample is excluded RFLP- Gaps Gaps: High quality of DNA is needed High quantity of DNA is needed Tedious process Length fragments vs. discrete alleles Strengths: Highly discriminating Established protocols Courtroom acceptance Polymerase Chain Reaction (PCR) Small amts of DNA (0.5-1 ng or about 152 diploid cells) Degraded DNA (small targeted regions) First tests not discriminating as RFLP 4

5 10/23/14 PCR Evidence Post ConvictionExoneration 270 cases individuals released from jail due to analysis of DNA evidence. 75% of original convictions were based on eye witness testimony 5

6 Extraction Quantitation Amplification of STR loci of interest Analysis /Interpretation Separation by CE CODIS Loci 11/7/12 Forensic DNA Profile D8 D21 D7 CSF D3 TH01 D13 D16 D2 D19 vwa TPOX D18 Amelogenin D5 FGA Identifiler 6

7 ABO Typing ABO Typing 7

8 Hair Bones Teeth Body Fluids Mitochondrial DNA (MtDNA) Useful for Hair, Teeth Bones 8

9 Only Mom contributes Mitochondrial DNA The Combined DNA Index System (CODIS) US DNA Database The DNA Identification Act of 1994 authorized the use of DNA data for forensic analysis and formalized CODIS. First and only Federal legislation concerning forensic science. Established quality standards and provided federal funding to assists State agencies Each State has passed DNA legislation concerning the collection of DNA samples from their citizens. 9

10 Database Hierarchy NDIS DNA Indices NDIS August 2013 The National Database (NDIS) has: 10,535,300 convicted offender profiles 509,900 forensic casework As of August 2013, CODIS has produced over 219,700 hits assisting in more than 210,700 investigations 10

11 Pennsylvania SDIS June ,372 offender profiles (up from 292,646) 12,132 casework profiles (up from 10,912) 5,442 investigations aided (up from 4,862) Note: there are 3 CODIS labs in PA (CA has 20 CODIS labs, NJ only has 2 CODIS labs, and CT has only one CODIS lab) Capability Gaps for hdna For law enforcement, national defense or national security laboratories, include the following: Non pristine evidentiary samples = mixtures Limited DNA quantities = low copy number DNA Variability in quality = degraded DNA Ability to develop investigative leads when there is no sample for comparison Can a NGS approach help us with these gaps? Mixtures In the old days, mixtures were more clearly resolvable as the detec4on sensi4vity was much lower Maybe 5% of the cases were truly complex As we ve enhanced the sensi4vity of our tests and started looking at items like touch evidence (LCN PCR), the number of cases with complex interpreta4on issues has risen significantly As high as 25% of the cases 11

12 Clean, Single Source Forensic DNA Profile Simple Mixture SWGDAM (Scientific Working Group on DNA Analysis Methods) Mission Statement The Scientific Working Group on DNA Analysis Methods, known as SWGDAM, serves as a forum to discuss, share, and evaluate forensic biology methods, protocols, training, and research to enhance forensic biology services as well as provide recommendations to the FBI Director on quality assurance standards for forensic DNA analysis. 12

13 Interpreting Mixtures and Low Copy DNA 2010 SWGDAM Guidelines Every DNA Laboratory should have a mixture interpretation section in their DNA protocol Example of a Low Level Minor Mixture?? Low Template Profile 13

14 Rela4ve Peak Height Intensi4es Technically, the peak height (intensity) of homozygote peaks should be twice the height of the heterozygote peaks Factors that Cause PHR Imbalance Heterozygous Peak Imbalance The imbalance may be due to a number of factors - stochas4c effects e.g., Reduced amounts of star/ng template Primer binding site anomalies/muta/ons Inhibitors that are disrup/ng the amplifica/on process; i.e., poor amplifica/on condi/ons Analy4cal Threshold Instrumental Response detec4on of DNA fragments versus instrument noise Analy1cal Threshold minimum peak height (intensity) necessary to iden4fy and differen4ate poten4al true alleles from instrument noise 14

15 Stochas4c Threshold Stochas1c Threshold minimum peak height (intensity) necessary to reduce (eliminate) the possibility of allelic dropout of the second allele of a heterozygote profile Has also been referred to as the Match Interpretation Threshold (MIT) Stochas4c Threshold 50 RFU Therefore, the stochastic threshold can be set at the same level as the analytical threshold for heterozygote profiles S/A Thresholds 100 RFU for Homozygotes 50 RFU for Heterozygotes Different thresholds for heterozygote and homozygote profiles 15

16 10/23/14 DNA as a Witness FORENSIC SCIENCE Capability Gap >50% Unsolved Cases The National Database (NDIS) has: 10,535,300 convicted offender profiles 509,900 forensic casework As of August 2013, CODIS has produced over 219,700 hits assisting in more than 210,700 investigations. What about the 200,000 cases still not solved? Forensically Relevant SNPs Kayser M, de Knijff P. Improving human forensics through advances in genetics, genomics and molecular biology. Nat. Rev. Genet. 2011;12(3): SNPs for: Identity-testing Lineage informative Ancestry informative Phenotype informative Budowle B, van Daal A. Forensically relevant SNP classes. Biotechniques. 2008;44(5):603 8, 610. Available at:

17 10/23/14 HIRIS Plex HIRIS Plex is an example of an early forensic method to get a phenotypic "snapshot from evidentiary DNA. It includes 24 SNPs that can predict eye color and hair color. FORENSIC SCIENCE HIRIS SNaPshot Multiplex System single-base extension DNA fragments separated by CE Uses GeneScan -120 LIZ Size Standard to size fragments. GeneMapper Software used to analyze the data and generate allele calls. FORENSIC SCIENCE 23andME 23andMe uses a system to type SNPs that provide ancestry information. You send in a vial of saliva and get up to 600,000 SNPs analyzed. This requires a lot of DNA! FORENSIC SCIENCE 17

18 Forensically Useful? Both methods provide valuable information, but there are some issues to consider when implementing these methods on forensic samples: 23andMe needs a tablespoon of saliva HIRIS Plex only provides a few SNPs FORENSIC SCIENCE Smith Team NGS Research } We will focus on the final gap: Ability to develop investigative leads when there is no sample for comparison } Using Next-Gen sequencing approach we will demonstrate the ability to interrogate more hdna variability, by examining single nucleotide polymorphisms (SNPS). MiSeq Pla^orm from Illumina 18

19 23andME Results FORENSIC SCIENCE 23andME Results 52 apsnps were typed by 23andMe 1 not concordant (rs182549) TT by 23andMe C/T by MiSeq 65 isnps were typed by 23andMe 1 not concordant (rs ) GG by 23andMe T/G by MiSeq FORENSIC SCIENCE Bang for the Buck (From 1 ng of DNA) 19

20 Illumina UAS prediction Internal Valida4on SWGDAM Valida4on Guidelines The internal valida4on process shall include the studies detailed below. If conducted within the same laboratory, developmental valida4on studies may sa4sfy some elements of the internal valida4on guidelines. The laboratory should evaluate the appropriate sample number and type, based on the methodology and/or applica4on necessary to demonstrate the poten4al limita4ons and reliability. The laboratory should determine the suitability of each study based on the methodology and may determine that a study is not necessary. Approved Dec,