1. DNA Polymorphisms and Human Identification
Chapter 11
DNA Polymorphisms and Human Identification

2. 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.

3. 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.

4. 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.

5. 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

6. Restriction Fragment Length Polymorphisms
Restriction fragment sizes are altered by changes in or between enzyme recognition sites.
GTCCAGTCTAGC
GAATTC
GTGGCAAAGGCT
CAGGTCAGATCG
CTTAAG
CACCGTTTCCGA
GAA A TC CG
TGGC C
AGGCT
CTT T AG GC
ACCG G
TCCGA
Point mutations GA
AGCGA
ATTC
GTGGC
AAAGGCT CT
TCGCT
TAAG
CACCG
TTTCCGA
Insertion (duplication)
Fragment insertion (or deletion)
GTTCTAGC
AAA
GGCT
GTGG
TCAGATCG
TTT
CCGA
CACC
AAAAAA
TTTTT T

7. Restriction Fragment Length Polymorphisms
The presence of RFLP is inferred from changes in fragment sizes.
Restriction site
Polymorphism
Gel band
pattern

8. Restriction Fragment Length Polymorphisms
The presence of RFLP is inferred from changes in fragment sizes. 1 2 A B C
Fragments visualized + -
(B+C)
(A+B)
(A+B+C)
Genotype I
++/+-
B, (B+C) II
+-/-+
(A+B), (B+C)
III
++/--
B,(A+B+C)
Probe
+/+ +/- -/+ -/-
Southern blot
band patterns
I II III

9. Restriction Fragment Length Polymorphisms
RFLP genotypes are inherited.
For each locus, one allele is inherited from each parent.
Southern blot
band patterns

10. Parentage Testing by RFLP
Which alleged father’s genotype has the paternal alleles?
AF 1
AF Mother
Locus
Locus
Locus
Child
Locus

11. 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
Locus Locus Locus 3

12. 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

13. Short Tandem Repeat Polymorphisms
STR alleles can be analyzed by fragment size (Southern blot).
One repeat unit
Allele
M M
Restriction site
Allele 1
GTTCTAGC
GGCC
GTGGC
AGCTAGCTAGCTAGCT
GCTG
GGCC
GTGG
CAAGATCG
CCGG
CACCG
TCGATCGATCGATCGA
CGAC
CCGG
CACC
tandem repeat
Allele 2
GTTCTAGC
GGCC
GTGGC
AGCTAGCTAGCT
GCTG
GTGG
CAAGATCG
CCGG
CACCG
TCGATCGATCGA
CGAC
CACC

14. Short Tandem Repeat Polymorphisms
STR alleles can also be analyzed by amplicon size (PCR).
(Genotype)

15. Short Tandem Repeat Polymorphisms
Allelic ladders are standards representing all alleles observed in a population.
11 repeats
5 repeats
(Allelic ladder)
Genotype: 6,8
Genotype: 7,9

16. Short Tandem Repeat Polymorphisms
Multiple loci are genotyped in the same reaction using multiplex PCR.
Allelic ladders must not overlap in the same reaction.

17. Short Tandem Repeat Polymorphisms by Multiplex PCR
FGA
TPOX
D8S1179
vWA
PentaE
D18S51
D2S11
THO1
D3S1358

18. 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

19. Analysis of Short Tandem Repeat Polymorphisms by PCR
STR genotypes are analyzed using gel or capillary gel electrophoresis.
11 repeats
5 repeats
11 repeats repeats
(Allelic ladder)
Genotype: 7,9

20. STR-PCR STR genotypes are inherited.
One allele is inherited from each parent.
Child’s alleles
Mother’s alleles
Father’s alleles

21. Parentage Testing by STR-PCR
Which alleged father’s genotype has the paternal alleles?

22. 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
AL S1 S2 V E M
AL S1 S2 V E M
Locus Locus Locus 3

23. 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.

24. Chimerism Testing Using STR
Allogeneic bone marrow transplants are monitored using STR.
Recipient
receives his or her own purged cells
receives donor
cells
Autologous
transplant
Allogeneic
A recipient with donor marrow is a chimera.

25. 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

26. Chimerism Testing Using STR: Informative Analysis
STR are scanned to find informative loci (donor alleles differ from recipient alleles).
Which loci are informative?
Locus:

27. 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.

28. Examples of Informative Loci (Type 5) [Thiede et al
Examples of Informative Loci (Type 5) [Thiede et al., Leukemia 18:248 (2004)]
Recipient
Stutter
Donor
Recipient
Donor

29. Examples of Noninformative Loci (Type 1)
Recipient
Donor

30. Chimerism Testing Using STR: Informative Analysis
Which loci are informative?
vWA TH Amel TPOX CSF1PO

31. 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) = area under recipient-specific peaks
A(D) = area under donor-specific peaks
A(D)
A(R)
A(R) + A(D)

32. Chimerism Testing Using STR: Engraftment Analysis
Formula for calculation of % recipient or % donor (no shared alleles).
A(R)
% Recipient DNA =
× 100
A(R) + A(D)
A(D)
% Donor DNA =
× 100
A(R) + A(D)

33. Chimerism Testing Using STR: Engraftment Analysis
Calculate % recipient DNA in post-1 and post-2:
Use the area under these peaks to calculate percentages.

34. 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.

35. 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

36. Chimerism Analysis of Cellular Subsets
Detection of different levels of engraftment in cellular subsets is split chimerism.
R D T G
R = Recipient alleles
D = Donor alleles
T = T-cell subset (mostly recipient) G = Granulocyte subset (mostly donor)

37. 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.

38. SNP Detection by Sequencing
T/T T/A A/A
5′ AGTCTG
5′ AG(T/A)CTG
5′ AGACTG

39. SNP Haplotypes
SNPs are inherited in blocks or haplotypes.

40. 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.

41. Mitochondrial DNA Polymorphisms
Sequence differences in the hypervariable regions (HV) of the mitochondrial genome.

42. 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.

43. 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.

44. 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.