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Fundamentals of Forensic
DNA Typing
http://nobelprize.org/nobel_prizes/literature/laureates/1938/buck.jpg
Pearl Buck
(Biology)
High RFLP
Multi-Locus Probes
Multiplex STRs
Power of RFLP
Discrimination Single Locus Probes
(Genetics)
PolyMarker
D1S80
mtDNA single STR
DQ
ABO
Low blood groups
Slow Fast
Speed of Analysis
(Technology)
Inheritance Patterns of ABO Blood Groups
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 3.2
A,B,AB, A,B, or
Fathers Blood Type
A A or O
or O AB
A or O
A,B,AB, A,B, or
B or O
B or O
AB
B or O Childs
Blood
A,B, or A,B, or A,B, or
AB AB AB AB
A or B Type
O A or O B or O A or B O
Advantages Limitation
Advantage Limitations
Probe b
a b e
d
c
d
a
e
Originally developed
by Alec Jeffreys Probe 1 Probe 2 Probe 3
Sizing ladder
Restriction Restriction
site site
VNTR
probe
7 repeats
Large
allele
Small allele
probe
13 repeats
Small
allele
Large allele
Bands seen on
autoradiogram of
probed membrane
Restriction Enzyme Cut Sites
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 3.5
TGCAG ANTCTAACG
HinfI Cellmark and
ACGTCTNA GATTGC European labs
5 base cutter Cuts on average
every ~1024 bases
TGCACTGCA GTAACG
PstI ACGTG ACGTCATTGC
Lifecodes
Use of different cut sites by various labs meant that results could not be compared
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/clinton/lewinskydress.html
Restriction Fragment Length Polymorphism
(RFLP) with single locus VNTR probes
Advantages Limitations
1. Excellent powers of 1. Limited sensitivity (>50 ng to 500 ng
discrimination (1 in millions or required).
greater with four loci). 2. Time-consuming process (days to
2. Large number of alleles (20 to 30 weeks) that cannot be automated.
3. Not suitable with degraded DNA
bins) at each locus which samples due to high molecular
facilitates mixed-sample weight needed.
analysis. 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.
(colorless) precipitate
Nylon membrane
Allele 1 Allele 2
dot
Nominal allele Subtype allele
specific dots specific dots
1.2 All
1 2 3 4 C 1.1 1.3 1.3 but AMPLITYPETM
4 1.3 DQ-Alpha
AB AB BB AB BC
S dot Fairly sensitive but low power of discrimination due to few alleles
DQA1
Sample 1
Sample 2
Positive Control
41 41
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 3.8
34 34
31 31
30 30
29 29
28 28
27 27
26 26
25 25
24 24
23 23
22 22
21 21
20 20
19 19
18 18
17 17
16 16
14 14
D1S80
Advantages Limitations
1. Improved sensitivity compared 1. Large allele range making it
to RFLP because it uses PCR. difficult to multiplex with other
2. Many alleles which facilitates loci and giving rise to
mixed-sample analysis. preferential amplification of
3. Discrete allele calling possible smaller alleles.
using allelic ladder, which also 2. Poor power of discrimination as
simplifies statistical a single locus (1 in 50).
interpretation. 3. Allele dropout seen with highly
degraded DNA.
4. Gel separation and silver-stain
detection not amenable to
automation or high-throughput
sample processing.
Repeat region
GAGGACCACCAGGAAG
16 bp repeat unit
Repeat region
TCAT
4 bp repeat unit
AmpFLP and GenePrint STRs
available circa 1993 to 2003
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 3.4
Sample 1
Sample 2
Ladders Ladders
CSF1PO 14
Detection with
CTT Triplex
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 3.10
TPOX 12
11
10
9 Double-bands for each allele
8 due to separation and detection
of forward and reverse strands
from each PCR product
TH01 11
10
9
8
7
6
5
Silver-Stained STRs
Advantages Limitations
1. Sensitive due to PCR. 1. Because only a single color
2. Relatively rapid process (a day channel is available, multiplex
or two). amplification and detection is
3. Works well with degraded DNA limited to 3 to 4 loci
samples since shorter 2. Both strands of DNA are
fragments of DNA can be detected leading to double
analyzed (compared to bands with some loci that can
D1S80). complicate interpretation.
4. A lower start-up cost compared
to fluorescent STRs
D3S1358 vWA
Amel D8S1179 TH01
D21S11 D16S539 D18S51
D19S433 D2S1338 SGM Plus
FGA
Fluorescent STR Profiles
from Two Individuals Using the SGM Plus Kit
John M. Butler (2005) Forensic DNA Typing, 2nd Edition, Figure 1.3
gender B C D
E H
ID
A F I
J
G
gender B
ID
A
E F
C H I
G J
D
Commercial STR 16plex Kits
SRM 2391b component 1
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.
Pentanucleotide Evaluation
Low Stutter -- High Heterozygosity
P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 P
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.
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
Potential Pitfalls with PCR Methods
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
http://www.bioteach.ubc.ca/MolecularBiology/DNAfingerprint/
The Past
RFLP
STRs
The Present
http://www.manastungare.com/publications/genetic/dna.gif
The DNA Field Moves Forward
The Future
Chapter 3 Points for Discussion
Why were single locus probes preferred over multi-locus
probes for RFLP testing?