chapter 11 dna polymorphisms and human identification
TRANSCRIPT
Chapter 11Chapter 11
DNA Polymorphisms and Human Identification
ObjectivesObjectives
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.
PolymorphismPolymorphism
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 SequencesPolymorphic 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 Types of Polymorphic DNA SequencesSequences
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 Restriction Fragment Length PolymorphismsPolymorphisms
Restriction fragment sizes are altered by changes in or between enzyme recognition sites.
GTCCAGTCTAGCGAATTCGTGGCAAAGGCTCAGGTCAGATCG CTTAAGCACCGTTTCCGA
GTCCAGTCTAGCGAAATCCGTGGCCAAGGCTCAGGTCAGATCG CTTTAGGCACCGGTTCCGA
Point mutationsGTCCAGTCTAGCGAAGCGAATTCGTGGCAAAGGCTCAGGTCAGATCG CTTCGCTTAAGCACCG TTTCCGA
Insertion (duplication)
Fragment insertion (or deletion)
GTTCTAGCGAATTCGTGGCAAA GGCTGAATTCGTGGTCAGATCGCTTAAGCACCGTTTCCGACTTAAGCACC
GTTCTAGCGAATTCGTGGCAAAAAA GGCTGAATTCGTGGTCAGATCG CTTAAGCACCGTTTTT TCCGACTTAAGCACC
Restriction Fragment Length Restriction Fragment Length Polymorphisms Polymorphisms
The presence of RFLP is inferred from changes in fragment sizes.
1 2
A B C
+ -AGATCT ATATCTTCTAGA TATAGA
1 2 Size Number+ + A,B,C 3+ - A, (B+C) 2- + (A+B), C 2- - (A+B+C) 1
+/+ +/- -/+ -/-
Polymorphism
Restriction site
Gel bandpattern
Restriction Fragment Length Restriction Fragment Length Polymorphisms Polymorphisms
The presence of RFLP is inferred from changes in fragment sizes.
Probe
Southern blot band patterns
1 2
A B C
1 2 Fragments visualized+ + B+ - (B+C)- + (A+B)- - (A+B+C)
Genotype Fragments visualizedI ++/+- B, (B+C)II +-/-+ (A+B), (B+C)III ++/-- B,(A+B+C)
+/+ +/- -/+ -/-
I II III
Restriction Fragment Length Restriction Fragment Length Polymorphisms Polymorphisms
RFLP genotypes are inherited. For each locus, one allele is inherited from each
parent. Father Mother Locus Locus 1 2 1 2
Parents
Child
Locus 1 2
Southern blot band patterns
Parentage Testing by RFLPParentage Testing by RFLP
Which alleged father’s genotype has the paternal alleles?
AF 1
AF2 Mother Locus Locus Locus 1 2 1 2 1 2
Child Locus
1 2
Evidence Testing by RFLPEvidence 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 Short Tandem Repeat Polymorphisms (STR)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 Short Tandem Repeat PolymorphismsPolymorphisms
STR alleles can be analyzed by fragment size (Southern blot).
GTTCTAGCGGCCGTGGCAGCTAGCTAGCTAGCTGCTGGGCCGTGGCAAGATCGCCGGCACCG TCGATCGATCGATCGACGACCCGGCACC
One repeat unit
tandem repeat
GTTCTAGCGGCCGTGGCAGCTAGCTAGCTGCTGGGCCGTGGCAAGATCGCCGGCACCG TCGATCGATCGACGACCCGGCACC
Restriction site
Allele 1
Allele 2
AlleleM 1 2 M
Short Tandem Repeat Short Tandem Repeat PolymorphismsPolymorphisms
STR alleles can also be analyzed by amplicon size (PCR).
....TCATTCATT CATT CATT CATTCATT CAT....
....AGTAAGTAAGTAAGTAAGTAAGTAAGTA....
....TCAT TCATT CATTCATT CATT CATTCATTCAT....
....AGTAAGTAAGTAAGTAAGTAAGTAAGTAAGTA....
Allele 1
Allele 2
PCR products:Allele 1 187 bp (7 repeats)Allele 2 191 bp (8 repeats)
7/8
(Genotype)
Short Tandem Repeat Short Tandem Repeat PolymorphismsPolymorphisms
Allelic ladders are standards representing all alleles observed in a population.
(Allelic ladder)
11 repeats
5 repeats
Genotype: 7,9 Genotype: 6,8
Short Tandem Repeat Short Tandem Repeat PolymorphismsPolymorphisms
Multiple loci are genotyped in the same reaction using multiplex PCR.
Allelic ladders must not overlap in the same reaction.
Short Tandem Repeat Short Tandem Repeat Polymorphisms by Multiplex PCRPolymorphisms by Multiplex PCR
FGA
TPOX
D8S1179
vWA
PentaE
D18S51
D2S11
THO1
D3S1358
AmelogeninAmelogenin Locus,Locus, HUMAMEL 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 Analysis of Short Tandem Repeat Polymorphisms by PCRPolymorphisms by PCR
STR genotypes are analyzed using gel or capillary gel electrophoresis.
Genotype: 7,9
11 repeats
5 repeats11 repeats 5 repeats
(Allelic ladder)
STR-PCRSTR-PCR
STR genotypes are inherited.
One allele is inherited from each parent.
Child’s alleles
Mother’s alleles
Father’s alleles
Parentage Testing by STR-PCRParentage Testing by STR-PCR
Which alleged father’s genotype has the paternal alleles?
Locus Child Mother 1 2D3S1358 16/17 16 17 17vWA 14/18 16/18 14/15 16/17FGA 21/24 20/21 24 24TH01 6 6/9.3 6/9 6/7TPOX 10/11 10/11 8/11 8/9CSF1PO 11/12 12 11 11/13D5S818 11/13 10/11 13 9/13D13S317 9/12 9 12/13 11/12
Evidence Testing by STR-PCREvidence 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 1 Locus 2 Locus 3
Short Tandem Repeat Short Tandem Repeat Polymorphisms: Y-STRPolymorphisms: 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 STRChimerism Testing Using STR
Allogeneic bone marrow transplants are monitored using STR.
Autologous transplant
Allogeneictransplant
Recipientreceives his or her own purged cells
Recipient receives donor cells
A recipient with donor marrow is a chimera.
Chimerism Testing Using STRChimerism Testing Using STR
There are two parts to chimerism testing: pretransplant informative analysis and post-transplant engraftment analysis
Before transplantDonor Recipient
After transplant
Complete (Full) Mixed Graft failure
Chimerism Testing Using STR:Chimerism Testing Using STR:Informative AnalysisInformative Analysis
STR are scanned to find informative loci (donor alleles differ from recipient alleles).
Which loci are informative?
M D R D R D R D R D R
Locus: 1 2 3 4 5
Chimerism Testing Using STR:Chimerism Testing Using STR:Informative AnalysisInformative 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.
Donor
Recipient
Recipient
Donor
Stutter
Examples of Informative LociExamples of Informative Loci (Type 5) [Thiede et al., Leukemia 18:248 (2004)](Type 5) [Thiede et al., Leukemia 18:248 (2004)]
Donor
Recipient
Examples of Noninformative Loci Examples of Noninformative Loci (Type 1)(Type 1)
vWA TH01 Amel TPOX CSF1PO
Chimerism Testing Using STR:Chimerism Testing Using STR:Informative AnalysisInformative Analysis
Which loci are informative?
Chimerism Testing Using STR:Chimerism Testing Using STR:Engraftment AnalysisEngraftment 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)
Chimerism Testing Using STR:Chimerism Testing Using STR:Engraftment AnalysisEngraftment Analysis
Formula for calculation of % recipient or % donor (no shared alleles).
% Recipient DNA = A(R) + A(D)
A(R) × 100
% Donor DNA = A(R) + A(D)
A(D) × 100
Chimerism Testing Using STR:Chimerism Testing Using STR:Engraftment AnalysisEngraftment Analysis
Calculate % recipient DNA in post-1 and post-2:
Use the area under these peaks to calculate percentages.
Chimerism Analysis of Cellular Chimerism Analysis of Cellular SubsetsSubsets
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 Chimerism Analysis of Cellular SubsetsSubsets
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
R D T G
R = Recipient allelesD = Donor allelesT = T-cell subset (mostly recipient)G = Granulocyte subset (mostly donor)
Chimerism Analysis of Cellular Chimerism Analysis of Cellular SubsetsSubsets
Detection of different levels of engraftment in cellular subsets is split chimerism.
Single Nucleotide Polymorphisms Single Nucleotide Polymorphisms (SNP)(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.
5′ AGTCTG 5′ AG(T/A)CTG 5′ AGACTG
T/T T/A A/A
SNP Detection by SequencingSNP Detection by Sequencing
haplotype~10,000 bp
SNP HaplotypesSNP Haplotypes
SNPs are inherited in blocks or haplotypes.
Applications of SNP AnalysisApplications 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.
HV 1(342 bp)
HV 2(268 bp)
Mitochondrial genome16, 600 bp
PL
PH1
PH2
Mitochondrial DNA Mitochondrial DNA PolymorphismsPolymorphisms
Sequence differences in the hypervariable regions (HV) of the mitochondrial genome.
Mitochondrial DNA Mitochondrial DNA PolymorphismsPolymorphisms
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.
SummarySummary
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.
SummarySummary
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.