Fundamentals of Forensic DNA Typing - PowerPoint PPT Presentation

Loading...

PPT – Fundamentals of Forensic DNA Typing PowerPoint presentation | free to view - id: 708a5f-ODQxM



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

Fundamentals of Forensic DNA Typing

Description:

Title: Fundamentals of Forensic DNA Typing Author: John M. Butler Last modified by: John M. Butler Created Date: 6/29/2009 11:19:37 PM Document presentation format – PowerPoint PPT presentation

Number of Views:82
Avg rating:3.0/5.0
Slides: 25
Provided by: John1633
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: Fundamentals of Forensic DNA Typing


1
Fundamentals of Forensic DNA Typing
Chapter 15 Additional Loci Non-Human DNA
  • Slides prepared by John M. Butler
  • June 2009

2
Chapter 15 Additional STR Loci, SNPs, and
Non-Human DNA
  • Chapter Summary
  • While a common set of core STR markers (e.g.,
    the 13 CODIS STRs) are widely used in human
    identity testing, additional STR markers and
    other genetic loci, such as single nucleotide
    polymorphisms (SNPs), will likely play a role in
    future applicationsparticularly when kinship
    analysis may be needed to resolve potential
    relatives from one another. In some cases,
    ancestry informative markers (AIMs) can be useful
    in estimating the ethnic origin of a sample. As
    additional information is gleaned from genome
    studies, genetic markers for physical traits may
    be discovered to enable phenotypic evaluation.
    Non-human DNA analysis has benefited from use of
    STR markers found in cats, dogs, and plants.

3
A 26plex Multiplex PCR Assay Developed at NIST
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 15.1
4
Alu Element Insertion PCR Assay
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, D.N.A. Box 15.2
5
Status of Genetic Marker Systems Used in
Forensic DNA Testing
  • STRs widely used in casework and national
    databases world-wide
  • miniSTRs smaller versions of STR loci that can
    work well on degraded DNA
  • Y-STRs permits examination of male-only DNA
  • mtDNA used in specialty labs for highly
    degraded specimens or hair that contains limited
    amounts of DNA
  • SNPs potential for identifying ethnicity of
    evidence sample still in research and likely to
    be limited in use

6
Single Nucleotide Polymorphisms (SNPs)
http//www.dna.gov/training/otc/
  • OTC Statement While the future utility of SNPs
    is uncertain, it seems unlikely that this method
    will replace the standard set of STRs used for
    routine DNA analysis due to the limited variation
    of SNPs and difficulties with mixed sample
    interpretation.
  • Butler, J.M., Coble, M.D., Vallone, P.M. (2007)
    STRs vs SNPs thoughts on the future of forensic
    DNA testing. Forensic Science, Medicine and
    Pathology 3 200-205.

7
More Possible Combinations Improved Ability to
Detect Mixtures
  • SNPs few possibilities
  • STRs many possibilities

Alleles
Alleles
A
B
A
B
C
D
E
F
Combinations
Combinations
AA AB AC AD AE AF
BB BC BD BE BF
CC CD CE CF
DD DE DF
EE EF
FF
AA
AB
BB
8
Why SNPs Will Likely Not Replace STRs
  • Large databases containing STR information (would
    need to replace data on existing samples with new
    DNA markers)
  • Mixture detection and interpretation benefits
    from marker systems with many alleles (SNPs only
    have two alleles and three genotype
    possibilities)
  • Degraded DNA can be successfully analyzed in many
    cases by miniSTRs (thus removing the primary
    motivation for using SNPs)

9
Compromised Sample Improvements
miniSTRs improve the success rate and recovery of
information from compromised DNA evidence
10
Approaches for challenging samples
perspectives for the future
  • Limited sample material (highly degraded DNA)
  • mtDNA (in use for this purpose since mid-1990s
    due to high copy number per cell)
  • Mixed male-female DNA
  • Y-chromosome STRs
  • Degraded DNA
  • miniSTRs
  • SNPs (?)

Chapter 10 in Forensic DNA Typing, 2nd Edition
http//www.cstl.nist.gov/biotech/strbase/y_strs.ht
m
http//www.cstl.nist.gov/biotech/strbase/miniSTR.h
tm
http//www.cstl.nist.gov/biotech/strbase/SNP.htm
11
A MiniFiler Kit Profile
Commercial miniSTR test from ABI focusing on
reducing PCR products length for 8 of the larger
Identifiler loci
Data from Becky Hill (NIST)
Applied Biosystems Kit
12
Further work with miniSTRs
NC01
NC02
We are currently developing tests for 26
additional miniSTR loci Potential utility for
missing persons work, kinship analysis, paternity
testing, mutation rates etc
Data from Becky Hill (NIST)
13
Comprised Sample Improvements (CSI) Conclusions
  • Analysis of shorter regions of DNA benefits
    recovery of information from degraded specimens
  • miniSTRs are now viewed as the primary way
    forward and a commercial kit is under development
  • SNPs, while theoretically beneficial due to small
    possible amplicons, are limited due to poor
    abilities to handle mixtures and the need for
    large multiplexes to improve powers of
    discrimination
  • mtDNA due to higher copy number per cell than
    nuclear DNA will continue to be used where
    limited samples are recovered (e.g., hair shafts
    and bone fragments)

14
Protocol Steps for Allele-Specific Primer
Extension SNP Assay
Genomic DNA sample
(Multiplex) PCR
ExoSAP Digestion
Amplification
Add SNP primer(s) and SNaPshot mix
SNP Extension (cycle sequencing)
SAP treatment
Primer Extension
Data Analysis (GeneScan)
Sample prep for 310/3100
Run on ABI 310/3100
Analysis
Add GS120 LIZ size standard
Use E5 filter (5-dye) and POP4 standard conditions
Type Sample (Genotyper or GeneMapperID)
15
Biogeographical Ancestry
  • Shriver, M.D. et al. (2003) Skin pigmentation,
    biogeographical ancestry and admixture mapping.
    Hum. Genet. 112(4)387-99
  • From abstract Ancestry informative markers
    (AIMs) are genetic loci showing alleles with
    large frequency differences between populations.
    AIMs can be used to estimate biogeographical
    ancestry at the level of the population, subgroup
    (e.g. cases and controls) and individual. This
    work indicates that it is possible to estimate
    the individual ancestry of a person based on DNA
    analysis with a reasonable number of well-defined
    genetic markers.

16
Biogeographical Ancestry (2)
  • Mark Shrivers work on ancestry informative
    markers has been commercialized through the
    company DNAPrint Genomics
  • http//www.dnaprint.com
  • http//www.ancestrybydna.com
  • Used in Derrick Todd Lee (Louisiana serial
    killer) case to overcome faulty eyewitness
    testimony of a Caucasian perpetrator

17
Pigmentation (Skin Color, etc.) Prediction
  • Lamason, R.L. et al. (2005) SLC24A5, a putative
    cation exchanger, affects pigmentation in
    zebrafish and humans. Science 3101782-1786
  • From abstract Lighter variations of pigmentation
    in humans are associated with diminished number,
    size, and density of melanosomes, the pigmented
    organelles of melanocytes. The variant allele is
    nearly fixed in European populations, is
    associated with a substantial reduction in
    regional heterozygosity, and correlates with
    lighter skin pigmentation in admixed populations,
    suggesting a key role for the SLC24A5 gene in
    human pigmentation.

18
Approximate Age Determination
  • Alvarez, M. and Ballantyne, J. (2006) The
    identification of newborns using messenger RNA
    profiling analysis. Anal. Biochem. 357(1)21-34.
  • From abstract In theory, it may be possible to
    determine patterns of gene expression that are
    age specific, thereby permitting the distinction
    among tissue samples originating from individuals
    of different ages (e.g., newborn, adolescent,
    middle-age, elderly). We have discovered two
    novel isoforms of gamma hemoglobin messenger RNA,
    designated HBG1n and HBG2n, which exhibit an
    extremely restricted pattern of gene expression,
    being confined to newborn individuals. Multiplex
    quantitative reverse transcription PCR (qRT-PCR)
    assays incorporating these novel mRNAs have been
    designed, tested, and evaluated for their
    potential forensic use. The results indicate that
    the assays provide the ability to determine
    whether a bloodstain originated from a newborn.

19
Age of Bloodstain Deposition
  • Anderson, S., Howard, B., Hobbs, G.R., Bishop,
    C.P. (2005) A method for determining the age of a
    bloodstain. Forensic Sci. Int. 148(1)37-45
  • From abstract If there were independent evidence
    that the biological sample was deposited at the
    time of the crime, then its age would reveal when
    the crime occurred. If the time of the crime were
    known through another means, then the age of the
    biological sample could potentially exclude the
    human source as a suspect. We have used real-time
    reverse transcriptase PCR to show that the ratio
    between different types of RNA (mRNA versus rRNA)
    changes over time in a linear fashion when dried
    human blood from eight individuals was examined
    over the course of 150 days.

20
Non-Human DNA Testing
  • Cat DNA
  • Dog DNA
  • Other uses of non-human DNA
  • Plant DNA for possible linkage to crime
    location
  • Marijuana DNA tracking drug sources
  • Animal as
  • Victim (abuse case)
  • Perpetrator (dog bite)
  • Silent witness (crime scene linkage)

21
New DNA Test for Cats Developed at NIST
J. Forensic Sci. 2005 50(5) 1061-1070
SRY (male)
7
Male cat
4
1
5
10
9
2
3
6
8
11
4
female
7
Female cat
2
5
10
6
1
9
8
3
11
22
SRY (male)
7
Male cat
4
1
5
10
9
2
3
6
8
11
4
7
Female cat
2
5
10
6
1
9
8
3
11
23
Animal DNA Testing
  • Most non-human DNA tests are specialty tests that
    will be rarely used by public labs and thus
    typically will be performed through outsourcing
    to a contract lab
  • QuestGen Forensics
  • http//www.questgen.biz/
  • UC Davis Vet Gen Lab
  • http//www.vgl.ucdavis.edu/

24
Challenges with Presenting Non-Human DNA in Court
(or other novel DNA methods)
Sensabaugh and Kaye (1998) Jurimetrics 38 1-16
  • Novelty of the application
  • Validity of the underlying scientific theory
  • Validity of any statistical interpretations
  • Relevant scientific community to consult in
    assessing the application may be limited
  • New methods may not have undergone the scientific
    scrutiny of regular forensic human DNA testing
    techniques

25
Chapter 15 Points for Discussion
  • Why are SNPs being considered for use in human
    identity testing?
  • What are the advantages and disadvantages of SNPs
    compared to currently used STR markers?
  • Will SNPs replace STRs as a primary means of
    forensic DNA testing? Why or why not?
  • Are there ethical challenges with using SNPs to
    predict ethnicity and physical traits? If so,
    what are they and how should the law enforcement
    community use this type of information in the
    future?
About PowerShow.com