Objectives Compare and contrast different types of polymorphisms

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 ATTCGTGGCAAAGGCT CAGGTCAGATCG CTTCGCTTAAG CACCG TTTCCGA Insertion (duplication) GTTCTAGCGAATTC GTGGCAAA GGCTGAATTC GTGG TCAGATCG CTTAAG CACCGTTTCCGACTTAAG CACC GTTCTAGCGAATTC 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 Polymorphism 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+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. Southern blot band patterns

Short Tandem Repeat Polymorphisms (STR) • STR are repeats of nucleotide sequences. • AAAAAA… - mononucleotide • ATATAT… - dinucleotide • TAGTAGTAG… - trinucleotide • TAGTTAGT… - tetranucleotide • TAGGCTAGGC… - pentanucleotide • Different alleles contain different numbers of repeats. • TTCTTC - four repeat allele • TTCTTCTTC - 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 GTTCTAGCGGCCGTGGCAGCTAGCTGCTGGGCCGTGG CAAGATCGCCGGCACCG TCGATCGACGACCCGGCACC tandem repeat Allele 2 GTTCTAGCGGCCGTGGCAGCTAGCTGCTGGGCCGTGG CAAGATCGCCGGCACCG TCGATCGACGACCCGGCACC

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

Short Tandem Repeat Polymorphisms by Multiplex PCR FGA Penta. E TPOX D 18 S 51 D 2 S 11 D 8 S 1179 THO 1 v. WA D 3 S 1358

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

Evidence Testing by STR-PCR Which suspect—S 1 or S 2—was at the crime scene? (V = victim, E = crime scene evidence, AL = Allelic ladder) AL S 1 S 2 V E M Locus 1 AL S 1 S 2 V E M Locus 2 AL S 1 S 2 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.

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.

Applications of SNP Analysis • SNPs can be used for mapping genes, human identification, chimerism analysis, and many other applications. • The Human Haplotype Mapping (Hap. Map) 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.

Mitochondrial DNA Polymorphisms • Mitochondria are maternally inherited. • There 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.