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 A TCCGTGGCCAAGGCT CAGGTCAGATCG CTTTAGGCACCG GTTCCGA
Point mutations
GTCCAGTCTAGC GA AGCGA ATTCGTGGCAAAGGCT CAGGTCAGATCG CTTCGCTTAAG CACCG TTTCCGA
Insertion (duplication)
Fragment insertion (or deletion)
GTTCTAGCGAATTC GTGGCAAA GGCTGAATTC GTGG
TCAGATCG CTTAAG CACCG TTTCCGA CTTAAG CACC
GTTCTAGCGAATTC GTGGCAAAAAA GGCTGAATTC GTGG
TCAGATCG CTTAAG CACCG TTTTT TCCGA CTTAAG CACC
Restriction Fragment Length Polymorphisms
The presence of RFLP is inferred from changes in fragment sizes.
1 2
A B C
+ -
AGATCT ATATCT
TCTAGA 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 band
pattern
Restriction Fragment Length 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 visualized
I ++/+- B, (B+C)
II +-/-+ (A+B), (B+C) III ++/-- B,(A+B+C)
+/+ +/- -/+ -/-
I II III
Restriction Fragment Length Polymorphisms
• RFLP genotypes are inherited.
• For each locus, one allele is inherited from each parent.
Father MotherLocus Locus 1 2 1 2
Parents
Child
Locus 1 2
Southern blot
band patterns
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).
GTTCTAGCGGCCGTGGCAGCTAGCTAGCTAGCTGCTGGGCCGTGG CAAGATCGCCGGCACCG TCGATCGATCGATCGACGACCCGGCACC
One repeat unit
tandem repeat
GTTCTAGCGGCCGTGGCAGCTAGCTAGCTGCTGGGCCGTGG CAAGATCGCCGGCACCG TCGATCGATCGACGACCCGGCACC Restriction site
Allele 1
Allele 2
Allele
M 1 2 M
Short Tandem Repeat Polymorphisms
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
Polymorphisms by Multiplex PCR
FGA
TPOX
D8S1179
vWA
PentaE
D18S51
D2S11
THO1
D3S1358
Parentage Testing by STR-PCR
Locus Child Mother 1 2
D3S1358 16/17 16 17 17
vWA 14/18 16/18 14/15 16/17 FGA 21/24 20/21 24 24
TH01 6 6/9.3 6/9 6/7
TPOX 10/11 10/11 8/11 8/9
CSF1PO 11/12 12 11 11/13
D5S818 11/13 10/11 13 9/13
D13S317 9/12 9 12/13 11/12
Which alleged father’s genotype has the paternal alleles?
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 1 Locus 2 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.
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.
5′ AGTCTG 5′ AG(T/A)CTG 5′ AGACTG
T/T T/A A/A
SNP Detection by Sequencing
haplotype
~10,000 bp
SNP Haplotypes
SNPs are inherited in blocks or haplotypes.
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.
HV 1
(342 bp) HV 2 (268 bp)
Mitochondrial genome 16, 600 bp
PL PH1 PH2
