Polymorphism
-seqeunce difference compared to standard
-single bases or thousands
-may or may not have phenotypic effects
-found througout the genome
-if location of polymorphic sequence is known it can serve as a marker for finding other genes
-polymorphic markers have different alleles
can be silent or outside of exons
Types of Polymorphic Sequences
*Restriction fragment length polymorphism (RFLP) - difference recognized by restriction enzyme at a specific site
- Variable-number tandem repeat (VNTR)
-10 to >50 base pairs - Short tandem repeat (STR) 1 - 10 base pairs
- Single-nucleotide polymorphism (SNP - one base
Restriction Fragment Length
Polymorphism (RFLP)
What is it
–when restriction fragments sizes are changed in or between enzyme recognition sites
-can be determined by the changes in fragment sizes and number
-during testing the RFLP genotypes are inherited one allele from each parent from each locus
-gel or capillary electrophoresis
-locus is position of chromosome
the plus is a recognition site
so A+B+C is 3 sites
A, B+C is 2 sites
Short Tandem Repeats (STR)
Polymorphism
what is it
-repeats of nucleotide sequences mono, di, tri
-different alleles can have different number of repeats
-can be analyzed by pcr - forensic
-allelic ladders are standards which represent all alleles represented in a population
-can use a multiplex PCR to genotype multiple loci in the same reaction but the allelic ladders should not overlap
-can be analyzed by pcr cge with gel (bands) or capillary electrophoresis (peaks)
-used for parentage testing, forensics, and engraftment after allogeneic bone marrow transplants
-two bands heterozygous if homo then one band
-finite repeats
for parent testing
-STR genotypes are inherited, one allele from each parent - AMEL amelogenin locus (used to determine the sex of the individual). The gene is located at 212 bp on x-mom(homo xx one band) and 218 bp at y-dad (hetero xy- two bands)
Forensic Testing by STR-PCR
-13 “core” loci as the Combined DNA Indexing System (CODIS)
-collection of alleles in all the loci tested
-Genetic concordance: all locus/alleles genotypes (alleles) from two sources (for example, suspect and crime evidence) are the same
1/n = likelihood of two unrelated individuals sharing the same genotype
STR Polymorphisms: Y-STR
-Y chromosome is inherited from father to
son without recombination
* STR’s on the Y chromosome are inherited paternally as a haplotype and used for exclusion and paternal lineage analysis
Mitochondrial DNA Polymorphisms
- Mitochondria are maternally inherited with differences on hypervariable regions (HV) averaging 8-9 bp differences
-maternal relatives will have the same mitochondrial differences and can be used for legal exclusion and confirmation of maternal linage
Engraftment Testing Using STR
-allogenic marrow transplants are best for cancer treatments
-autologous patients own cells
-allogenic from another person
-a recipient with donor marrow is a chimera
-marrow transplants monitored using STR
-STR scan to find informative loci
-donor alleles differ from recipient allele
-where bands or peaks dont overlap
-stutter in donor bands/peaks should be avoided
-a stutter is technical artifact from PCR reaction where small amount of repeat units is produced
-post transplant the peak areas (A) in informative loci are determined in fluorescence units
A-R recipient
A-D donor
look at equation
Single-Nucleotide Polymorphisms (SNP)
- Single-nucleotide differences among DNA sequences
- One SNP occurs every ~1,000 base pairs in human DNA
- detected by genome sequencing, melt-curve analysis,
- 99% have no biological effect
- inherited in blocks or haplotypes
*can be used for gene mapping, human identification, chimerism analysis
*sickle cell example glu-val - The Human Haplotype Mapping (HapMap) Project identified SNP haplotypes throughout the human genome
