Chapter 3

Historical Methods
Fundamentals of Forensic
DNA Typing

Slides prepared by John M. Butler

June 2009
 Value of a Historical Review

If you want to understand

today, you have to search
yesterday.

http://nobelprize.org/nobel_prizes/literature/laureates/1938/buck.jpg
Pearl Buck

Attributed to Pearl Buck

(http://www.quotegarden.com/history.html)

The Nobel Prize

in Literature 1938
 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-DQ 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.
 Comparison of DNA Typing Technologies
Markers Used
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 3.1

(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

Mothers Blood Type

A B AB O

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

4 possible types: A, B, AB, and O

 Blood Group Typing

Advantages Limitation

1. Rapid, simple tests. 1. Poor power of discrimination (1

2. Only test available for many in 10) with such few alleles (i.e.,
years. inclusions are not very
meaningful).

John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 3.6

 Forensic Protein Profiling
Isoenzyme Characteristics
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 3.1

Erythrocyte Protein Number of Discrimination

Isoenzyme Symbol Alleles Potential

Phosphoglucomutase PGM 2 10 phenotypes

(4 alleles with possible with
IEF) IEF
Erythrocyte Acid ACP/EAP 3 0.67
Phosphatase

Esterase D ESD 2 0.27

Adenylate Kinase AK 2 0.25

Glyoxalase I GLO 2 0.57

Adenosine ADA 2 0.06

Deaminase
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.
 Forensic Protein Profiling

Advantage Limitations

1. Improved power of 1. Poor power of discrimination (1

discrimination over blood group in 100) even with multiple
typing. 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

 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
 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
 VNTRs
HaeIII HaeIII HaeIII HaeIII
Size Separation on Gel
a b c d e
Probe
VNTR repeat region
c

Probe b
a b e
d
c
d
a
e

Only DNA fragments containing

a complementary sequence to
the probe are detected
 Single-Locus
Multi-Locus Probe
Probe
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 3.3

Originally developed
by Alec Jeffreys Probe 1 Probe 2 Probe 3

Probe 33.6 D1S7 D2S44 D4S139

Complex patterns Better for forensic samples containing mixtures
Multi-Locus Probe RFLP Results
Are More Complicated to Interpret
Restriction Enzyme Cut Sites
Electrophoresis of DNA Fragments
Transfer of DNA Fragments to a Nylon
Membrane - Southern Blotting
 Probing of Membrane with
Radioactive or Labeled DNA Probes
Exposure of Labeled Membrane
to Autoradiographic Film
Nomenclature for Autoradiograms
Repeated Probing of Same Membrane
to Yield a Series of Autoradiograms

Sequential Detection of RFLP Single Locus Probes

 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

Chromosome VNTR Number Repeat Unit

Heterozygosity HaeIII Fragment
Designation Probe of Bins Size (kb) Length (bp)

D1S7 MS1 28 0.945 0.5-12 9

D2S44 yNH24 26 0.926 0.7-8.5 31
D4S139 pH30 19 0.899 2-12 31
D10S28 pTBQ7 24 0.943 0.4-10 33
D14S13 pCMM101 30 0.899 0.7-12 15
D17S79 V1 19 0.799 0.5-3 38
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.
 Key Processes with RFLP Analysis
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 3.4

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

TGCAGG CCTAACG FBI, RCMP, U.S.

HaeIII public labs
ACGTCC GGATTGC
4 base cutter Cuts on average
every ~256 bases

TGCAG ANTCTAACG
HinfI Cellmark and
ACGTCTNA GATTGC European labs
5 base cutter Cuts on average
every ~1024 bases

TGCACTGCA GTAACG
PstI ACGTG ACGTCATTGC
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
 DNA Evidence and Monica Lewinskys Blue Dress
John M. Butler (2009) Fundamentals of Forensic DNA Typing, D.N.A. Box 3.1

August 17, 1998

FBI Report on Analysis
of Stain on Monica
Lewinskys Blue Dress

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.

John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 3.6

John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 2.2

DNA Typing Methods

Comparison of RFLP and PCR-based
 Reverse Dot Blot Detection
TMB Colored
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 3.6

(colorless) precipitate

Relies on hybridization of sample

PCR products to test allele dots
HRP
Strepavidin

Biotin GTCCAGTCG 3 PCR product (denatured)

hybridization
CAGGTCAGC 5 Immobilized SSO probe

Nylon membrane

Allele 1 Allele 2

View from above

nylon membrane
match no match
 Reverse Dot Blot Tests
(a) Control AmpliType DQ
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 3.7

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

(b) AmpliType PM + DQA1 (PolyMarker)

1.2 All
4.2
1 2 3 4 C 1.1 1.3 1.3 but 4.1
4.3
DQA1 1.2/3
4 1.3

LDLR GYPA HBGG D7S8 GC

S A B A B A B C A B A B C

AB AB BB AB BC

S dot Fairly sensitive but low power of discrimination due to few alleles
 DQA1

C control dot to test that sample

is above stochastic threshold
Image from Elseviers Encyclopedia of Forensic Sciences
 AmpliType PM (PolyMarker)

S dot to test that sample is above stochastic threshold

Image from Elseviers Encyclopedia of Forensic Sciences

 Characteristics of DQA1 and PolyMarker Loci
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 3.3

Chromosomal Number PCR Product

Locus Gene Product Location of Alleles Heterozygosity Size

DQA1 HLA-DQA1 6p21.3 7 0.828 242/239 bp

LDLR Low density 19p13.1- 2 0.493 214 bp
lipoprotein 13.3
receptor
GYPA Glycophorin A 4q28-31 2 0.498 190 bp
HBGG Hemoglobin G 11p15.5 3 0.508 172 bp
gammaglo
bin
D7S8 -- 7q22-31.1 2 0.476 151 bp
GC Group-specific 4q11-13 3 0.592 138 bp
component
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.
 DQA1 + PM
Advantages Limitations
1. Fast, simple method (compared 1. Poor power of discrimination (1
to RFLP). in 1000) with only six loci
2. Capable of analyzing small or developed each containing only
degraded samples because it a few alleles.
uses PCR. 2. Mixture interpretation difficult
3. No instrumentation needed after with limited number of alleles
PCR. per locus.

John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 3.6

 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
 D1S80 Gel Image

Image from Elseviers Encyclopedia of Forensic Sciences

 Allelic Allelic
Ladder Ladder

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.

John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 3.6

 Minisatellite Marker (D1S80)
Flanking regions
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 3.9

Repeat region

GAGGACCACCAGGAAG
16 bp repeat unit

STR Marker (TH01)

Flanking regions

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

Kit/Locus PCR product Chromosomal Repeat Number of Heterozygosity

sizes Location Size Alleles

D1S80 369-801 bp 1p 16 bp >30 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

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
 Allelic Allelic
Silver-Stain

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

John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 3.6

 Early fluorescent STR multiplex systems
available during the 1990s
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 3.5

Assay/Kit Lab or Supplier STR Locus

British Home Office Forensic Science Service TH01, VWA, F13A1,
Quadruplex FES/FPS
Second Generation Forensic Science Service TH01, VWA, FGA, D8S1179,
Multiplex (SGM) 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
 PCR Product Size (bp)
Different STR Kits Run on the
D3S1358
Same DNA Sample
vWA FGA
Blue
Different combinations
of STR loci exist
TH01 TPOX CSF1PO
Amel
Green I

D3S1358 TH01 D13S317 CSF1PO

Amel D5S818 vWA TPOX FGA D7S820 Profiler

Amel D8S1179 vWA D13S317

D7S820
D3S1358 D5S818 D21S11 FGA Profiler Plus
D18S51

D3S1358 TH01 D7S820

TPOX
Amel D16S539
CSF1PO COfiler

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

D8 Identifiler kit (Applied Biosystems)

multiplex STR result
TH01
D13
D19 D5 VWA
TPOX D16
AMEL D21 CSF
D3 FGA
D18 D2
D7

PowerPlex 16 kit (Promega Corporation)

multiplex STR result
D8
D18 CSF Penta E
D5 D21
AMEL D7
VWA
D3 TH01 D13 FGA Penta D
TPOX D16

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

24 samples from different individuals

Slide courtesy of Jim Schumm, Bode Technology Group

 Fluorescent STRs
Advantages Limitations
1. Sensitive due to PCR (single cell analysis has 1. Less discrimination power per
been demonstrated). locus compared to VNTRs due
2. Relatively rapid process (can be completed in a to a smaller number of alleles
few hours or at most a day or two). and less heterozygosity per
3. Works fairly well with degraded samples since
shorter fragments of DNA can be analyzed
locus.
(compared to D1S80). miniSTRs have extended 2. The possibility of contamination
the capabilities for degraded sample analysis. from stray DNA is increased
4. Multiplex PCR amplification and multi-color because of the PCR
fluorophore labeling and detection enables amplification process.
examining 15 or more loci simultaneously, which 3. Expensive equipment required
provide excellent powers of discrimination (1 in for detection.
billions or greater with 8 to 9 or more STR loci) 4. Stutter products and unbalanced
5. Standardized sets of core loci are widely used
with availability of commercial STR kits. peak heights may occur and
6. Automated detection enables high-throughput make the interpretation of
sample processing. mixtures more difficult.
7. The potential number of loci is very large, which 5. Data interpretation must account
is important if siblings or other relatives are for artifacts such as dye blob,
involved. electrophoretic spikes, etc.

John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 3.6

 DNA template 3-TAAATGATTCC-5
5 3
Primer A
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Figure 3.11

anneals AT Extension produces a series of

ATT ddNTP terminated products each
one base different in length
ATTT
ATTTA
ATTTAC Each ddNTP is labeled
ATTTACT with a different color
fluorescent dye
ATTTACTA
ATTTACTAA
ATTTACTAAG
ATTTACTAAGG

Sequence is read by noting peak

color in electropherogram
(possessing single base resolution)
 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
 Mitochondrial DNA Sequencing
Advantages
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.
John M. Butler (2009) Fundamentals of Forensic DNA Typing, Table 3.6
Limitations
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.
 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
 Potential Pitfalls with PCR Methods

1. The target DNA template may not amplify due to the

presence of PCR inhibitors in the extracted DNA.

2. 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.
 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
 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?

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?