Chapter 11
DNA Polymorphisms and Human Identification
Objectives
 Compare and contrast different types of
polymorphisms.
 Define restriction fragment length polymorphisms.
 Describe short tandem repeat structure and
nomenclature.
 Describe gender identification using the amelogenin
locus.
 Illustrate the use of STR for bone marrow engraftment
monitoring.
 Define single nucleotide polymorphisms.
 Discuss mitochondrial DNA typing.
Polymorphism
 A DNA polymorphism is a sequence
difference compared to a reference
standard that is present in at least 1–2%
of a population.
 Polymorphisms can be single bases or
thousands of bases.
 Polymorphisms may or may not have
phenotypic effects.
Polymorphic DNA Sequences
 Polymorphisms are found throughout the
genome.
 If the location of a polymorphic sequence
is known, it can serve as a landmark or
marker for locating other genes or
genetics regions.
 Each polymorphic marker has different
versions or alleles.
Types of Polymorphic DNA
Sequences
 RFLP: restriction fragment length
polymorphisms
 VNTR: variable number tandem repeats
(8 to >50 base pairs)
 STR: short tandem repeats (1–8 base
pairs)
 SNP: single-nucleotide polymorphisms
Restriction Fragment Length
Polymorphisms
Restriction fragment sizes are altered by changes in
or between enzyme recognition sites.
GTCCAGTCTAGC GAATTC GTGGCAAAGGCT
CAGGTCAGATCG CTTAAG CACCGTTTCCGA
GTCCAGTCTAGC GAA ATCCGTGGCCAAGGCT
CAGGTCAGATCG CTTTAGGCACCGGTTCCGA
Point mutations
GTCCAGTCTAGC GAAGCGA ATTC GTGGCAAAGGCT
CAGGTCAGATCG CTTCGCTTAAG CACCG TTTCCGA
Insertion (duplication)
GTTCTAGC GAATTC GTGGCAAA GGCTGAATTC GTGG
TCAGATCG CTTAAG CACCGTTTCCGACTTAAG CACC
GTTCTAGC GAATTC GTGGCAAAAAA GGCTGAATTC GTGG
TCAGATCG CTTAAG CACCGTTTTT TCCGACTTAAG CACC
Fragment insertion (or deletion)
Restriction Fragment Length
Polymorphisms
The presence of RFLP is inferred from
changes in fragment sizes.
Restriction site
+
-
AGATCT
TCTAGA
ATATCT
TATAGA
1
A
1
+
+
-
Polymorphism
2
B
2
+
+
-
C
Size
Number
A,B,C
3
+/+
A, (B+C)
2
(A+B), C
2
(A+B+C)
1
+/- -/+ -/-
Gel band
pattern
Restriction Fragment Length
Polymorphisms
The presence of RFLP is inferred from
changes in fragment sizes.
1
A
1
+
+
-
2
B
2
+
+
-
Genotype
I
++/+II
+-/-+
III
++/--
C
Fragments visualized
B
(B+C)
(A+B)
(A+B+C)
Fragments visualized
B, (B+C)
(A+B), (B+C)
B,(A+B+C)
Probe
+/+ +/- -/+ -/-
I
II III
Southern blot
band patterns
Restriction Fragment Length
Polymorphisms
 RFLP genotypes are inherited.
 For each locus, one allele is inherited from each
parent.
Father
Locus
1
2
Mother
Locus
1
2
Parents
Southern blot
band patterns
Locus
1
2
Child
Parentage Testing by RFLP
Which alleged father’s genotype has the
paternal alleles?
AF 1
Locus
1
2
AF2
Child
Locus
1
2
Locus
1
2
Mother
Locus
1
2
Evidence Testing by RFLP
Which suspect—S1 or S2—was at the crime scene?
(V = victim, E = crime scene evidence, M = molecular
weight standard)
M S1 S2 V E M
M S1 S2 V E M
M S1 S2 V E M
Locus 1
Locus 2
Locus 3
Short Tandem Repeat
Polymorphisms (STR)
 STR are repeats of nucleotide sequences.





AAAAAA… - mononucleotide
ATATAT… - dinucleotide
TAGTAGTAG… - trinucleotide
TAGTTAGTTAGT… - tetranucleotide
TAGGCTAGGCTAGGC… - pentanucleotide
 Different alleles contain different numbers of
repeats.
 TTCTTCTTCTTC - four repeat allele
 TTCTTCTTCTTCTTC - five repeat allele
Short Tandem Repeat
Polymorphisms
STR alleles can be analyzed by fragment
size (Southern blot).
One repeat unit
Allele
M 1 2 M
Restriction site
Allele 1
GTTCTAGCGGCCGTGGCAGCTAGCTAGCTAGCT GCTGGGCCGTGG
CAAGATCG CCGGCACCG TCGATCGATCGATCGA CGACCCGGCACC
tandem repeat
Allele 2
GTTCTAGCGGCCGTGGCAGCTAGCTAGCT GCTGGGCCGTGG
CAAGATCG CCGGCACCG TCGATCGATCGA CGACCCGGCACC
Short Tandem Repeat
Polymorphisms
STR alleles can also be analyzed by
amplicon size (PCR).
Allele 1
....TCATTCATT CATT CATT CATTCATT CAT....
....AGTAAGTAAGTAAGTAAGTAAGTAAGTA....
Allele 2
....TCAT TCATT CATTCATT CATT CATTCATTCAT....
....AGTAAGTAAGTAAGTAAGTAAGTAAGTAAGTA....
PCR products:
Allele 1 187 bp (7 repeats)
Allele 2 191 bp (8 repeats)
(Genotype)
7/8
Short Tandem Repeat
Polymorphisms
Allelic ladders are standards representing
all alleles observed in a population.
11 repeats
(Allelic ladder)
5 repeats
Genotype: 7,9
Genotype: 6,8
Short Tandem Repeat
Polymorphisms
 Multiple loci are genotyped in the same
reaction using multiplex PCR.
 Allelic ladders must not overlap in the
same reaction.
Short Tandem Repeat
Polymorphisms by Multiplex PCR
FGA
PentaE
TPOX
D18S51
D2S11
D8S1179
THO1
vWA
D3S1358
Amelogenin Locus, HUMAMEL
 The amelogenin locus is not an STR.
 The HUMAMEL gene codes for amelogenin-like
protein.
 The gene is located at Xp22.1–22.3 and Y.
X allele = 212 bp
Y allele = 218 bp
Females (X, X) - homozygous
Males (X, Y) - heterozygous
Analysis of Short Tandem Repeat
Polymorphisms by PCR
STR genotypes are analyzed using gel or
capillary gel electrophoresis.
11 repeats
11 repeats
5 repeats
5 repeats
(Allelic ladder)
Genotype: 7,9
STR-PCR
 STR genotypes are inherited.
Child’s alleles
Mother’s alleles
Father’s alleles
 One allele is inherited from each parent.
Parentage Testing by STR-PCR
Which alleged father’s genotype has the
paternal alleles?
Locus
D3S1358
vWA
FGA
TH01
TPOX
CSF1PO
D5S818
D13S317
Child
16/17
14/18
21/24
6
10/11
11/12
11/13
9/12
Mother
16
16/18
20/21
6/9.3
10/11
12
10/11
9
1
17
14/15
24
6/9
8/11
11
13
12/13
2
17
16/17
24
6/7
8/9
11/13
9/13
11/12
Evidence Testing by STR-PCR
Which suspect—S1 or S2—was at the crime scene?
(V = victim, E = crime scene evidence, AL = Allelic
ladder)
AL S1 S2 V E M
Locus 1
AL S1 S2 V E M
Locus 2
AL S1 S2 V E M
Locus 3
Short Tandem Repeat
Polymorphisms: Y-STR
 The Y chromosome is inherited in a block
without recombination.
 STR on the Y chromosome are inherited
paternally as a haplotype.
 Y haplotypes are used for exclusion and
paternal lineage analysis.
Chimerism Testing Using STR
Allogeneic bone marrow transplants are
monitored using STR.
Autologous
transplant
Allogeneic
transplant
Recipient
receives his or
her own purged
cells
Recipient
receives donor
cells
A recipient with donor marrow is a chimera.
Chimerism Testing Using STR
There are two parts to chimerism testing: pretransplant
informative analysis and post-transplant engraftment
analysis
Before transplant
Donor
Recipient
After transplant
Complete (Full)
Mixed
Graft failure
Chimerism Testing Using STR:
Informative Analysis
 STR are scanned to find informative loci
(donor alleles differ from recipient alleles).
 Which loci are informative?
Locus:
M
1
D
2
R
D
3
R
D
4
R
D
5
R
D
R
Chimerism Testing Using STR:
Informative Analysis
 There are different degrees of
informativity.
 With the most informative loci, recipient
bands or peaks do not overlap stutter in
donor bands or peaks.
 Stutter is a technical artifact of the PCR
reaction in which a minor product of n-1
repeat units is produced.
Examples of Informative Loci
(Type 5) [Thiede et al., Leukemia 18:248 (2004)]
Recipient
Stutter
Donor
Recipient
Donor
Examples of Noninformative Loci
(Type 1)
Recipient
Donor
Chimerism Testing Using STR:
Informative Analysis
Which loci are informative?
vWA
TH01
Amel
TPOX
CSF1PO
Chimerism Testing Using STR:
Engraftment Analysis
 Using informative loci, peak areas are
determined in fluorescence units or from
densitometry scans of gel bands.
A(R) + A(D)
A(D)
A(R)
 A(R) = area under recipient-specific peaks
 A(D) = area under donor-specific peaks
Chimerism Testing Using STR:
Engraftment Analysis
Formula for calculation of % recipient or %
donor (no shared alleles).
% Recipient DNA =
A(R)
A(R) + A(D)
× 100
% Donor DNA =
A(D)
A(R) + A(D)
× 100
Chimerism Testing Using STR:
Engraftment Analysis
Calculate % recipient DNA in post-1 and
post-2:
Use the area
under these
peaks to
calculate
percentages.
Chimerism Analysis of Cellular
Subsets
 Cell subsets (T cells, granulocytes, NK
cells, etc.) engraft with different kinetics.
 Analysis of cellular subsets provides a
more detailed description of the engrafting
cell population.
 Analysis of cellular subsets also increases
the sensitivity of the engraftment assay.
Chimerism Analysis of Cellular
Subsets
 T cells (CD3), NK cells (CD56),
granulocytes, myeloid cells (CD13,
CD33), myelomonocytic cells (CD14), B
cells (CD19), stem cells (CD34)
 Methods
 Flow cytometric sorting
 Immunomagnetic cell sorting
 Immunohistochemistry + XY FISH
Chimerism Analysis of Cellular
Subsets
Detection of different levels of engraftment
in cellular subsets is split chimerism.
R
D
T
R = Recipient alleles
D = Donor alleles
T = T-cell subset (mostly recipient)
G = Granulocyte subset (mostly donor)
G
Single Nucleotide Polymorphisms
(SNP)
 Single-nucleotide differences between
DNA sequences.
 One SNP occurs approximately every
1,250 base pairs in human DNA.
 SNPs are detected by sequencing, melt
curve analysis, or other methods.
 99% have no biological effect;
60,000 are within genes.
SNP Detection by Sequencing
T/T
5′ AGTCTG
T/A
5′ AG(T/A)CTG
A/A
5′ AGACTG
SNP Haplotypes
SNPs are inherited in blocks or haplotypes.
haplotype
~10,000 bp
Applications of SNP Analysis
 SNPs can be used for mapping genes,
human identification, chimerism analysis,
and many other applications.
 The Human Haplotype Mapping
(HapMap) Project is aimed at identifying
SNP haplotypes throughout the human
genome.
Mitochondrial DNA
Polymorphisms
Sequence differences in the hypervariable
regions (HV) of the mitochondrial genome.
HV 1
(342 bp)
PH1
PH2
HV 2
(268 bp)
PL
Mitochondrial genome
16, 600 bp
Mitochondrial DNA
Polymorphisms
 Mitochondria are maternally inherited.
 There are an average of 8.5 base
differences in the mitochondrial HV
sequences of unrelated individuals.
 All maternal relatives will have the same
mitochondrial sequences.
 Mitochondrial typing can be used for legal
exclusion of individuals or confirmation of
maternal lineage.
Summary
 Four types of polymorphisms are used for a
variety of purposes in the laboratory: RFLP,
VNTR, STR, and SNP.
 Polymorphisms are used for human
identification and parentage testing.
 Y-STR haplotypes are paternally inherited.
 Polymorphisms are used to measure
engraftment after allogeneic bone marrow
transplants.
Summary
 Single-nucleotide polymorphisms are
detected by sequencing, melt curve analysis,
or other methods.
 SNPs can be used for the same applications
as other polymorphisms.
 Mitochondrial DNA typing is performed by
sequencing the mitochondrial HV regions.
 Mitochondrial types are maternally inherited.