Vallone and Coble, Potential Application of Forensic Methods to Cancer Research February 17, 2006 http:///www.cstl.nist.gov/biotech/strbase/NISTpub.htm 1 Potential Application of Forensic DNA Testing Methods to Cancer Diagnostics Peter M. Vallone and Michael D. Coble National Institute of Standards and Technology, Gaithersburg, MD February 17,2006 Fred Hutchinson Cancer Research Center Seattle,WA •STR loci and kits •Real Time PCR for DNA quantitation •SNP markers •Case example •Interlaboratory Studies •NIST Standard Reference Materials (SRMs) •miniSTRs •Sample Quality Control Outline Location of NIST (Gaithersburg, MD) Washington D.C. Dulles Airport Reagan National Airport BWI Airport NIST FBI Lab Baltimore, MD Richmond, VA Capitol Beltway (I-495) I-270 I-95 I-95 I-66 ~30 miles * Biochemical Science Division DNA Measurements Human Identity Project (NIJ Funded) A Brief History of Forensic DNA Typing 1985 1990 1994 1996 1998 2000 2002 1992 UK National Database launched (April 10, 1995) 2006: DNA is an important part of the criminal justice system 2004 2006 Capillary electrophoresis of STRs first described First STRs developed First commercial fluorescent STR multiplex kits U.S. core loci defined STR typing with CE is routinely used worldwide PCR developed Y-chromosome STRs STR = short tandem repeat mtDNA Gill et al. (1985) Forensic application of DNA 'fingerprints‘. Nature 318:577-9 US National Database launched (October 13, 1998) 13 CODIS Core STR Loci with Chromosomal Positions D5S818 D21S11 TH01 TPOX D13S317 D16S539 D18S51 D8S1179 D3S1358 VWA AMEL AMEL Tetra nucleotide repeats Probability of a random match: ~1 in 594 trillion Commercial PCR primer kits exist for typing these loci Forensic DNA Testing •Probe subsets of genetic variation in order to differentiate between individuals •DNA typing must be done efficiently and reproducibly (information must hold up in court) •Typically, we are not looking at genes – little/no information about race, predisposal to disease, or phenotypical information (eye color, height, hair color) is obtained
16
Embed
Vallone and Coble, Potential Application of February 17 ... · Vallone and Coble, Potential Application of Forensic Methods to Cancer Research February 17, 2006
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Vallone and Coble, Potential Application of Forensic Methods to Cancer Research
Potential Application of Forensic DNA Testing Methods to Cancer Diagnostics
Peter M. Vallone and Michael D. CobleNational Institute of Standards and Technology, Gaithersburg, MD
February 17,2006Fred Hutchinson Cancer Research Center
Seattle,WA
•STR loci and kits
•Real Time PCR for DNA quantitation
•SNP markers
•Case example
•Interlaboratory Studies
•NIST Standard Reference Materials (SRMs)
•miniSTRs
•Sample Quality Control
Outline
Location of NIST (Gaithersburg, MD)
Washington D.C.
Dulles Airport
Reagan National Airport
BWI Airport
NIST
FBI Lab
Baltimore, MD
Richmond, VA
Capitol Beltway(I-495)
I-270I-95
I-95
I-66
~30 miles*
Biochemical Science Division
DNA Measurements
Human Identity Project(NIJ Funded)
A Brief History of Forensic DNA Typing
1985
1990
1994 1996
1998 2000
2002
1992
UK National Database launched
(April 10, 1995)
2006: DNA is an important part of the criminal justice system
2004
2006
Capillary electrophoresis of STRs first describedFirst STRs
developed
First commercial fluorescent STR multiplex kits
U.S. core loci defined
STR typing with CE is routinely used worldwide
PCR developed
Y-chromosome STRs
STR = short tandem repeat
mtDNA
Gill et al. (1985) Forensic application of DNA 'fingerprints‘. Nature 318:577-9
US National Database launched (October 13, 1998)
13 CODIS Core STR Loci with Chromosomal Positions
CSF1PO
D5S818
D21S11
TH01
TPOX
D13S317
D7S820
D16S539 D18S51
D8S1179
D3S1358
FGAVWA
AMEL
AMEL
Tetra nucleotide repeats
Probability of a random match:~1 in 594 trillion
Commercial PCR primer kits existfor typing these loci
Forensic DNA Testing
•Probe subsets of genetic variation in order to differentiate between individuals
•DNA typing must be done efficiently and reproducibly (information must hold up in court)
•Typically, we are not looking at genes – little/no information about race, predisposal to disease, or phenotypical information (eye color, height, hair color) is obtained
Vallone and Coble, Potential Application of Forensic Methods to Cancer Research
To date: (>100,000 allele calls)Identifiler (15 autosomal markers + Amelogenin) (10,608)Roche Linear Arrays (HV1/HV2 10 regions) (6,630)Y STRs 22 loci—27 amplicons (17,388)Y STRs 27 new loci (14,535)Yfiler kit 17 loci (11,237)Y SNPs 50 markers on sub-set of samples (11,498)Orchid 70 autosomal SNPs on sub-set (13,230)miniSTR testing-new loci and CODIS concordance (9,228)New miniSTR loci – for 11 loci, 7,293 genotypesmtDNA full control region sequences by AFDIL
DNA extracted from whole blood (anonymous; self-identified ethnicities) received from Interstate Blood Bank (Memphis, TN) and Millennium Biotech Inc. (Ft. Lauderdale, FL)
U.S. Population Dataset260 Caucasians, 260 African Americans, 140 Hispanics, 3 Asians = 663 males
Vallone, P.M., Decker, A.E., Butler, J.M. (2005) Allele frequencies for 70 autosomal SNP loci with U.S. Caucasian, African American, and Hispanic samples. Forensic Sci. Int. 149: 279-286.
Autosomal 12plex SNP Assay
RMP: ~1 in 300,000 (average ~1 in 70,000)
chr: 5 15 10 1 17 13 17 1 6 11 20 11
C/T
C/C
T/T
G/A
C/C C/CC/CC/T
C/TC/T T/TT/T
locus: 1 2 3 4 5 6 7 8 9 10 11 12
SNaPshot product nucleotide sizes (relative to GS120 LIZ)
“Best” 12 SNPs selected from 70 originally tested;>0.45 heterozygosity in U.S. Caucasians, Hispanics, and African Americans
Vallone, P.M., Decker, A.E., Butler, J.M. (2005) Allele frequencies for 70 autosomal SNP loci with U.S. Caucasian, African American, and Hispanic Samples. Forensic Sci. Int. 149:279-286.
12plex PCR and 12plex ASPE from 0.5 ng DNA template
Vallone, et al. (2005) Progress in Forensic Genetics 11, in press
Population data has been collected on >1,000 samples (662 U.S. and 375 world samples) from 10 different population groups
all PCR products <90 bp
Vallone and Coble, Potential Application of Forensic Methods to Cancer Research
12-plex PCR followed by 12-plex ASPEFragments separated on a ABI 3100 in 35 minutesThe 12-plex assay has been run on over 1000 samplesWorks well on 0.5 to 1 ng of template
32 cycle PCR1.5 U Taq Gold15 µL volume
RMP: ~1 in 300,000 (average ~1 in 70,000)
Ambiguous call panel 14a
TT-CT-CT-CT-TT-CTJK4022
Ambiguous call panel 12a
JK311CT-TT-CT-CT-CT-CT
Poor heterozygote balance makes conclusive typing difficult in some of the 6-plex assays
A1 B1
C1 D1
Panel 12a
35 samples indicated some level of peak imbalance
Overview of Sample Quality and Quantity Issues
• Autosomal STR typing experiments exhibited mixtures that may indicate well to well contamination. Some of the STR profiles indicated a 1:1 mixture ratio.
• In addition, qPCR human DNA quantitation experiments conducted at NIST indicate that the actual concentrations varied from 100 pg to almost 10 ng.
• The low concentration samples are a concern because of the possibility of stochastic imbalance during PCR (allele drop-in or drop-out) and thus potential unreliable SNP results.
Plate A1 qPCR
0.000.501.001.502.002.503.003.504.00
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76
Sample
ng/u
L
Expected concentration of DNA samples provided Alu-detection quantitative real-time
PCR assay for human DNA
Of the 80 samples examined in Plate A1 with the Alu qPCR human DNA quantitation assay,13 samples were observed with <100 pg/uL DNA rather than the expected 1-2 ng/uL based on the sample information provided.
Profiler Plus – Partial ProfileDegradation or Inhibition
12,392 Bone samples processed3,405 Full profiles (13 STR loci)2,143 High partial profiles (>7 STR loci)2,670 Low partial profiles (<7 STR loci)4,174 No loci
Over6800
profiles
Larger PCR products fail STR repeat region
miniSTR primer
miniSTR primer
Conventional PCR primer
Conventional PCR primer
Conventional STR test (COfiler™ kit)
MiniSTR assay (using Butler et al. 2003 primers)
A miniSTR is a reduced size STR amplicon that enables higher recovery of information from degraded DNA samples
Coble and Butler (2005) Characterization of new miniSTR loci to aid analysis of degraded DNA J. Forensic Sci. 50(1): 43-53
miniSTR Assay Sensitivity (D10S1248)
200 pg
100 pg
50 pg
20 pg
10 pg
5 pg
28 cycles – 1U Taq 32 cycles – 2U Taq
Sensitivity - Degraded DNA from an OU Bone Sample
10 pg/µL (30pg input DNA), 32 cycles, 2U Taq
Amelogenin
D3
D5
vWA
TH01
D13
D21
D8TPOX
D7Loss of larger alleles
PP16
Sensitivity - Degraded DNA from an OU Bone Sample
Amelogenin
D3
D5
vWA
TH01
D13
D21
D8TPOX
D7
NC01 NC02
D10
D22
D14
D1
D4
D2
Global Impact of NC miniSTRs FSI (2006) 156(2): 242-244
…recommended that existing multiplexes are re-engineered to enable small amplicon detection, and that three new mini-STR loci with alleles <130 bp(D10S1248, D14S1434 and D22S1045) are adopted as universal. This will increase the number of European standard Interpol loci from 7 to 10.
2) 8 instances of 100% complete mutational reversions affecting only haplogroup V diagnostic mutations AND a simultaneous gain of 31 completely homoplasmicmutations at only the diagnostic polymorphisms associated with the African haplogroup L3.
1) A sample switch occurred.
2) 8 instances of 100% complete mutational reversions affecting only haplogroup V diagnostic mutations AND a simultaneous gain of 31 completely homoplasmicmutations at only the diagnostic polymorphisms associated with the African haplogroup L3.
Decision“IT’S POSSIBLE”
STR Analysis of Blood and TumorSTR Marker Blood 09 Tumor 09Amelogenin X, X X, YD3S1358 16, 17 15, 16
Lessons Learned• Bandelt et al. (2004), Yao et al. (2004), Salas et al.
(2005) – practical suggestions for improved quality control and methods to identify errors in data using the well-established mtDNA phylogeny.
• Increased interactions with experts in the mtDNA world (aDNA, population genetics, etc…)
• Sample quality – LCN, degradation can lead to erroneous results (e.g. artifacts, contamination) – need for improved protocols (controls) and established methods for analyses.