Fundamentals of Forensic DNA Typing - PowerPoint PPT Presentation

Loading...

PPT – Fundamentals of Forensic DNA Typing PowerPoint presentation | free to download - id: 6adb42-ZjdlY



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:

Chapter 3 Historical Methods Fundamentals of Forensic DNA Typing Slides prepared by John M. Butler June 2009 – PowerPoint PPT presentation

Number of Views:77
Avg rating:3.0/5.0
Slides: 55
Provided by: JohnMB54
Learn more at: http://cstl.nist.gov
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 3 Historical Methods
  • Slides prepared by John M. Butler
  • June 2009

2
Value of a Historical Review
  • If you want to understand today, you have to
    search yesterday.
  • Attributed to Pearl Buck
  • (http//www.quotegarden.com/history.html)

Pearl Buck
http//nobelprize.org/nobel_prizes/literature/laur
eates/1938/buck.jpg
3
Chapter 3 Historical Methods
  • Chapter Summary
  • Over the past several decades, methods for DNA
    testing have improved in terms of sensitivity,
    speed, and strength of result. The first DNA
    tests involved Southern blotting of restriction
    fragment length polymorphisms (RFLPs) separated
    on agarose gels and detected with radioactive
    probes. RFLP techniques required substantial
    amounts of intact DNA and could take weeks to
    obtain results especially with single-locus
    probes. Early polymerase chain reaction (PCR)
    methods, such as HLA-DQa and D1S80, appeared in
    the early 1990s and improved sensitivity but were
    initially less informative than single locus or
    multi-locus RFLP methodologies. In the mid-1990s,
    short tandem repeat markers (STRs) became
    available in commercial kit formats, first as
    silver-stained monoplexes or simple multiplexes
    and eventually in multi-colored fluorescence
    detection megaplex assays that are widely used
    today. Simultaneously, mitochondrial DNA (mtDNA)
    sequencing was implemented in the forensic DNA
    community to aid recovery of information from
    samples containing limited or highly degraded DNA
    such as hair and skeletal remains.

4
Comparison of DNA Typing Technologies
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 3.1
5
Inheritance Patterns of ABO Blood Groups
Mothers Blood Type
A B AB O
A A or O A,B,AB, or O A,B, or AB A or O
B A,B,AB, or O B or O A,B, or AB B or O
AB A,B, or AB A,B, or AB A,B, or AB A or B
O A or O B or O A or B O
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 3.2
Childs Blood Type
Fathers Blood Type
4 possible types A, B, AB, and O
6
Blood Group Typing
Advantages Limitation
1. Rapid, simple tests. 2. Only test available for many years. 1. Poor power of discrimination (1 in 10) with such few alleles (i.e., inclusions are not very meaningful).
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Table 3.6
7
Forensic Protein Profiling Isoenzyme
Characteristics
Erythrocyte Isoenzyme Protein Symbol Number of Alleles Discrimination Potential
Phosphoglucomutase PGM 2 (4 alleles with IEF) 10 phenotypes possible with IEF
Erythrocyte Acid Phosphatase ACP/EAP 3 0.67
Esterase D ESD 2 0.27
Adenylate Kinase AK 2 0.25
Glyoxalase I GLO 2 0.57
Adenosine Deaminase ADA 2 0.06
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Table 3.1
Information from Li, R. (2008) Forensic Biology.
Boca Raton CRC Press, p. 169 and Ballantyne, J.
(2000) Serology in Encyclopedia of Forensic
Sciences. San Diego Academic Press.
8
Forensic Protein Profiling
Advantage Limitations
1. Improved power of discrimination over blood group typing. 1. Poor power of discrimination (1 in 100) even with multiple systems. 2. Poor sensitivity. 3. Proteins are not always stable in forensic stains or found in every sample.
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Table 3.6
9
Historical Methods for DNA Testing
  • RFLP
  • Multi-locus probes
  • Single-locus probes
  • PCR based sequence polymorphisms
  • HLA-DQ alpha
  • PM DQA1
  • AmpFLPs
  • D1S80
  • Silver-stained STRs
  • Fluorescently detected STRs

10
The Principles of RFLP Testing
  • Cut the DNA with biological scissors
    (Restriction enzymes)
  • Separate Fragments of differing Length by gel
    electrophoresis
  • Detect length-based differences (Polymorphisms)
    in DNA fragments of interest

11
VNTRs
HaeIII
HaeIII
HaeIII
HaeIII
Size Separation on Gel
a
b
c
d
e
VNTR repeat region
c
b
a
b
e
d
c
d
a
e
Only DNA fragments containing a complementary
sequence to the probe are detected
12
Originally developed by Alec Jeffreys
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 3.3
Complex patterns
Better for forensic samples containing mixtures
13
Multi-Locus Probe RFLP Results Are More
Complicated to Interpret
14
Restriction Enzyme Cut Sites
15
Electrophoresis of DNA Fragments
16
Transfer of DNA Fragments to a Nylon Membrane -
Southern Blotting
17
Probing of Membrane with Radioactive or Labeled
DNA Probes
18
Exposure of Labeled Membrane to Autoradiographic
Film
19
Nomenclature for Autoradiograms
20
Repeated Probing of Same Membrane to Yield a
Series of Autoradiograms
Sequential Detection of RFLP Single Locus Probes
21
Characteristics of Single-Locus VNTR Probes and
RFLP Markers Used in North American in the Late
1980s and 1990s
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Table 3.2
Adapted from Budowle, B. et al. (2000) DNA Typing
Protocols Molecular Biology and Forensic
Analysis. Natick, MA Eaton Publishing NIJ
(2000) The Future of Forensic DNA Testing
Predictions of the Research and Development
Working Group.
22
Key Processes with RFLP Analysis
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 3.4
23
Restriction Enzyme Cut Sites
HaeIII
FBI, RCMP, U.S. public labs
4 base cutter
Cuts on average every 256 bases
HinfI
Cellmark and European labs
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 3.5
5 base cutter
Cuts on average every 1024 bases
PstI
Lifecodes
6 base cutter
Cuts on average every 4096 bases
Use of different cut sites by various labs meant
that results could not be compared
24
DNA Evidence and Monica Lewinskys Blue Dress
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, D.N.A. Box 3.1
http//www.law.umkc.edu/faculty/projects/ftrials/c
linton/lewinskydress.html
25
Restriction Fragment Length Polymorphism (RFLP)
with single locus VNTR probes
Advantages Limitations
1. Excellent powers of discrimination (1 in millions or greater with four loci). 2. Large number of alleles (20 to 30 bins) at each locus which facilitates mixed-sample analysis. 1. Limited sensitivity (gt50 ng to 500 ng required). 2. Time-consuming process (days to weeks) that cannot be automated. 3. Not suitable with degraded DNA samples due to high molecular weight needed. 4. Essentially continuous allele sizes which requires grouping alleles into bins. Binning introduces statistical complications and sometimes difficulties of interpretation. 5. Limited number of validated loci (4 to 6 loci commonly used) which meant that these VNTRs were of limited value in distinguishing between siblings.
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Table 3.6
26
Comparison of RFLP and PCR-based DNA Typing
Methods
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Table 2.2
27
Reverse Dot Blot Detection
TMB (colorless)
Colored precipitate
Relies on hybridization of sample PCR products to
test allele dots
HRP
Strepavidin
PCR product (denatured)
Biotin
GTCCAGTCG
3
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 3.6
hybridization
28
Reverse Dot Blot Tests
AmpliType DQa
AmpliType PM DQA1 (PolyMarker)
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 3.7
Fairly sensitive but low power of discrimination
due to few alleles
29
DQA1
C control dot to test that sample is above
stochastic threshold
Image from Elseviers Encyclopedia of Forensic
Sciences
30
AmpliType PM (PolyMarker)
S dot to test that sample is above stochastic
threshold
Image from Elseviers Encyclopedia of Forensic
Sciences
31
Characteristics of DQA1 and PolyMarker Loci
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Table 3.3
Adapted from Budowle, B. et al. (2000) DNA Typing
Protocols Molecular Biology and Forensic
Analysis. Natick, MA Eaton Publishing NIJ
(2000) The Future of Forensic DNA Testing
Predictions of the Research and Development
Working Group.
32
DQA1 PM
Advantages Limitations
1. Fast, simple method (compared to RFLP). 2. Capable of analyzing small or degraded samples because it uses PCR. 3. No instrumentation needed after PCR. 1. Poor power of discrimination (1 in 1000) with only six loci developed each containing only a few alleles. 2. Mixture interpretation difficult with limited number of alleles per locus.
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Table 3.6
33
D1S80
  • AmpFLP amplified fragment length polymorphism
  • 16 bp repeat sequence
  • PCR products in 400-800 bp range
  • Separated on a polyacrylamide gel
  • Detected with silver staining
  • Sometimes combined with amelogenin to facilitate
    sex-typing

34
D1S80 Gel Image
Image from Elseviers Encyclopedia of Forensic
Sciences
35
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 3.8
36
D1S80
Advantages Limitations
1. Improved sensitivity compared to RFLP because it uses PCR. 2. Many alleles which facilitates mixed-sample analysis. 3. Discrete allele calling possible using allelic ladder, which also simplifies statistical interpretation. 1. Large allele range making it difficult to multiplex with other loci and giving rise to preferential amplification of smaller alleles. 2. Poor power of discrimination as a single locus (1 in 50). 3. Allele dropout seen with highly degraded DNA. 4. Gel separation and silver-stain detection not amenable to automation or high-throughput sample processing.
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Table 3.6
37
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 3.9
38
AmpFLP and GenePrint STRsavailable circa 1993 to
2003
Kit/Locus PCR product sizes Chromosomal Location Repeat Size Number of Alleles Heterozygosity
D1S80 369-801 bp 1p 16 bp gt30 0.82
TH01 179-203 bp 11p15.5 4 bp 8 0.76
TPOX 224-252 bp 2p23-2pter 4 bp 8 0.65
CSF1PO 295-327 bp 5q33.5-34 4 bp 10 0.75
F13A1 281-331 bp 6p24-25 4 bp 14 0.76
F13B 169-193 bp 1q31-q32.1 4 bp 6 0.75
LPL 105-133 bp 8p22 4 bp 10 0.77
FES/FPS 222-250 bp 15q25-qter 4 bp 6 0.69
vWA 131-171 bp 12p12-pter 4 bp 10 0.83
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Table 3.4
Heterozygosities from Caucasian database in
Creacy, S.D. et al. (1995) Proceedings of the
Sixth International Symposium on Human
Identification. Madison, Wisconsin Promega pp.
28-31
39
Silver-Stain Detection with CTT Triplex
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 3.10
40
Silver-Stained STRs
Advantages Limitations
1. Sensitive due to PCR. 2. Relatively rapid process (a day or two). 3. Works well with degraded DNA samples since shorter fragments of DNA can be analyzed (compared to D1S80). 4. A lower start-up cost compared to fluorescent STRs 1. Because only a single color channel is available, multiplex amplification and detection is limited to 3 to 4 loci 2. Both strands of DNA are detected leading to double bands with some loci that can complicate interpretation.
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Table 3.6
41
Early fluorescent STR multiplex systems available
during the 1990s
Assay/Kit Lab or Supplier STR Locus
British Home Office Quadruplex Forensic Science Service TH01, VWA, F13A1, FES/FPS
Second Generation Multiplex (SGM) Forensic Science Service TH01, VWA, FGA, D8S1179, D18S51, D21S11, amelogenin
CTTv Promega Corporation CSF1PO, TPOX, TH01, VWA
FFFL Promega Corporation F13A1, F13B, FES/FPS, LPL
GammaSTR Promega Corporation D16S539, D13S317, D7S820, D5S818
AmplSTR Blue Applied Biosystems D3S1358 VWA, FGA
AmplSTR Green Applied Biosystems CSF1PO, TPOX, TH01, amelogenin
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Table 3.5
42
Different STR Kits Run on the Same DNA Sample
Different combinations of STR loci exist
43
Fluorescent STR Profiles from Two Individuals
Using the SGM Plus Kit
John M. Butler (2005) Forensic DNA Typing, 2nd
Edition, Figure 1.3
44
Commercial STR 16plex Kits
SRM 2391b component 1
Identifiler kit (Applied Biosystems) multiplex
STR result
D8
TH01
D13
VWA
D19
D5
TPOX
D16
CSF
AMEL
D21
FGA
D3
D18
D2
D7
PowerPlex 16 kit (Promega Corporation)
multiplex STR result
D8
Penta E
D18
CSF
D5
D21
D7
AMEL
VWA
D13
Penta D
D3
TH01
FGA
D16
TPOX
From Butler, J.M. (2005) Constructing STR
multiplex assays. Methods in Molecular Biology
Forensic DNA Typing Protocols (Carracedo, A.,
ed.), Humana Press Totowa, New Jersey, 297
53-66.
45
Pentanucleotide Evaluation Low Stutter -- High
Heterozygosity
24 samples from different individuals
Slide courtesy of Jim Schumm, Bode Technology
Group
46
Fluorescent STRs
Advantages Limitations
1. Sensitive due to PCR (single cell analysis has been demonstrated). 2. Relatively rapid process (can be completed in a few hours or at most a day or two). 3. Works fairly well with degraded samples since shorter fragments of DNA can be analyzed (compared to D1S80). miniSTRs have extended the capabilities for degraded sample analysis. 4. Multiplex PCR amplification and multi-color fluorophore labeling and detection enables examining 15 or more loci simultaneously, which provide excellent powers of discrimination (1 in billions or greater with 8 to 9 or more STR loci) 5. Standardized sets of core loci are widely used with availability of commercial STR kits. 6. Automated detection enables high-throughput sample processing. 7. The potential number of loci is very large, which is important if siblings or other relatives are involved. 1. Less discrimination power per locus compared to VNTRs due to a smaller number of alleles and less heterozygosity per locus. 2. The possibility of contamination from stray DNA is increased because of the PCR amplification process. 3. Expensive equipment required for detection. 4. Stutter products and unbalanced peak heights may occur and make the interpretation of mixtures more difficult. 5. Data interpretation must account for artifacts such as dye blob, electrophoretic spikes, etc.
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Table 3.6
47
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Figure 3.11
48
mtDNA Sequencing
  • May be used when limited or highly degraded DNA
    is available such as with a hair shaft or bone
    specimen
  • Requires a great deal of labor and expertise and
    thus only a few forensic laboratories conduct
    mtDNA analysis
  • Due to maternal inheritance without
    recombination, siblings and maternal relatives
    will have identical mtDNA making it less useful
    in many human identity applications

49
Mitochondrial DNA Sequencing
Advantages Limitations
1. Excellent sensitivity and success with limited or badly damaged samples due to large number of mtDNA molecules per cell and the fact that the small mtDNA molecule is more robust than nuclear DNA. 2. Maternal transmission from a mother to all of her children extends possible reference sample providers and enables tracing family lineages. 3. A majority of mtDNA haplotypes occur only once in a population database making its power of discrimination better than a single nuclear locus. 1. Since all descendants through the female line are identical, mtDNA analysis cannot be used to distinguish among members of a sibship or maternal relatives. 2. Without recombination, the discrimination power of mtDNA is limited by the size of the population database used. 3. Heteroplasmy (the occurrence of more than one mtDNA type in a single cell or person) can complicate the analysis. 4. Because the entire mtGenome is inherited as a unit, it is equivalent to a single nuclear locus. Hence, the discrimination power is limited by the size of the database.
John M. Butler (2009) Fundamentals of Forensic
DNA Typing, Table 3.6
50
Advantages of PCR Methods
  • Overall, PCR-based methods have greatly enabled
    forensic DNA analysis for the following reasons
  • Very small amounts of DNA template may be used
    even as little as from a single cell.
  • DNA degraded to fragments only a few hundred base
    pairs in length can serve as effective templates
    for amplification.
  • Large numbers of copies of specific DNA sequences
    can be amplified simultaneously with multiplex
    PCR reactions.
  • Contaminant DNA, such as from fungal and
    bacterial sources, will not amplify because
    human-specific primers are used.
  • Commercial kits are now available for easy PCR
    reaction setup and amplification

51
Potential Pitfalls with PCR Methods
  • The target DNA template may not amplify due to
    the presence of PCR inhibitors in the extracted
    DNA.
  • Amplification may fail due to sequence mutations
    in the primer-binding region of the genomic DNA
    template something often referred to as a null
    allele.
  • 3. Contamination from other human DNA sources
    besides the forensic evidence at hand or
    previously amplified DNA samples is possible
    without the use of careful laboratory technique
    and validated protocols.

52
  • Report published in Nov 2000
  • Asked to estimate where DNA testing would be 2,
    5, and 10 years into the future
  • Conclusions
  • STR typing is here to stay for a few years
    because of DNA databases that have grown to
    contain millions of profiles

http//www.ojp.usdoj.gov/nij/pubs-sum/183697.htm
53
The DNA Field Moves Forward
The Future
The Past
The Present
http//www.bioteach.ubc.ca/MolecularBiology/DNAfin
gerprint/
STRs
RFLP
54
Chapter 3 Points for Discussion
  • Why were single locus probes preferred over
    multi-locus probes for RFLP testing?
  • Why did STRs replace RFLP as the method of choice
    in modern forensic DNA analysis?
  • Discuss the advantages and disadvantages of short
    tandem repeat (STR) markers relative to other
    potential genetic loci that could be used for DNA
    testing.
  • What is the difference between a mini-satellite
    and a micro-satellite?
About PowerShow.com