Microsatellites

What is microsatellite
Simple Sequence Repeats (SSR) 1-6 bp long

Classification of Microsatellites

Simple microsatelltes Composite microsatellites

(GT)n (AC)n (AG)n

Simple microsatellites contain only one kind of repeat sequences:

Composite microsatellites contain more than one type repeats

Molecular Basis of Microsatellite Polymorphism

Different by 3 repeats

Slippage of DNA polymerase is believed to be the major cause of microsatellite variation The mutation rate can be as high as 0.1 to 0.2% per generation

Abundant and Even Distribution

Abundant
Abundance varies with species, but all species studied to date have miocrosatellites In well studied mammal species, one microsatellite exist in every 30-40 kb DNA.

Even distribution
On all chromosomes On all segments of chromosomes With genes Often in introns In exons as well Trinucleotide repeats and human diseases: Huntington disease, fragile X, and other mental retardation-related human diseases

Small Locus sizes adapt them for PCR

PCR 2 6 3 1

Microsatellites are co-dominant markers

AB CD BC CC

AD

BC

BD

CD

AC

AB

BD

AC

BD

AB

Allele A

Allele B Allele C Allele D

Mendelian Inheritance of Microsatellites

Microsatellites are inherited as codominant markers according to Mendelian laws

Liu et al. 1999. Biochem. Biophys. Res Comm. 259: 190-194 Liu et al. 1999. J. Heredity 90: 307-311.

Advantages of Microsatellite Markers

Abundant

Evenly distributed

Highly polymorphic

Small loci

Co-dominant

Development of microsatellite markers

Need

SSR containing clones Sequences of the flanking regions of SSR

Microsatellites-enriched Small-insert DNA Libraries (I)

Genomic DNA Digest with several 4-bp blunt enders Gel fraction of 300-600 bp Ligation to a phagemid vector
insert
micro Small insert 3.5 kb

insert

insert

insert

Small insert Small insert 3.4 kb Small insert 3.4 kb Small insert 3.4 kb 3.4 kb

insert

insert

insert

insert

insert

Small insert 3.4 kb

Small insert 3.4 kb

Small insert 3.4 kb

Small insert 3.4 kb

Small insert 3.4 kb

Microsatellites-enriched Libraries (II)

micro
insert insert insert

in sert

Small insert plasmids 3.5 kb

Small insert plasmids 3.5 kb

Small insert plasmids 3.5 kb

Small insert plasmids 3.5 kb

Using dut/ungCJ236 strain u u u

Single-stranded phagemids 3.5 kb

Conversion into single-stranded phagemids using helper phage u micro u u u u u u u u u Single-stranded phagemids Single-stranded phagemids Single-stranded phagemids
3.5 kb 3.5 kb
3.5 kb

Wont be converted to ds will be degraded in WT host

Microsatellite-enriched Libraries (III)

micro

Convert into ds using (CA)15 (e.g.)

u micro ds plasmids

micro

Single-stranded phagemids 3.5 kb

u
3.5 kb 3.5 kb

Transform into WT E. coli

micro ds plasmids 3.5 kb

According to Ostrander et al., 1992: PNAS 89:3419

Microsatellites-enriched Libraries

CA GA TA CG CT GT

CAA CAT CAG CAC CGG CGT CGC CGA ...

4 bp

5 bp

Characterization of Microsatellites
Isolate plasmid DNA; sequence clones; Identify clones with enough sequences for primer design.

PCR Optimization and PIC Analysis

PCR products best <200 bp PCR conditions: annealing temperature, Mg++, pH, DMSO, etc. Polymorphism information content Polymorphism in reference families

Disadvantages of microsatellites
Previous genetic information is needed Huge Upfront work required Problems associated with PCR of microsatellites

The concept of Polymorphic information content

Measures the usefulness of a marker Informativeness in specific families

Microsatellite Genotyping

1. AA x AA 2. AA x BB 3. A x 4. AA x AB 5. AA x B 6. A x AB 7. AB x AB

Not polymorphic No segregation Only 1 allele segregating 1:1

B segregates 1:1, A segregates with intensity 1:1

A not segregate B segregates 1:1 A segregates 3:1, B segregates 1:1 A segregates 3:1, B segregates 3:1

Microsatellite Genotyping

8. A x B 9. AB x

A segregates 1:1, B segregates 1:1 A segregates 1:1, B segregates 1:1, A & B alternating 2 of the 3 alleles segregating 1:1 All 3 alleles segregating 1:1, 2 types with only 1 allele 2 of 3 alleles segregating 1:1, the other 3:1 with a single allele existing for some individuals All 4 alleles segregating 1:1

10. AA x BC

11. A x BC

12. AB x AC

13. AB x CD

Polymorphic Information Content PIC)

PIC refers to the value of a marker for detecting polymorphism within a population PIC depends on the number of detectable alleles and the distribution of their frequency. Bostein et al. (1980) Am. J. Hum Genet. 32:314331. Anderson et al. (1993). Genome 36: 181-186.

Polymorphic Information Content (PIC)

n PICi = 1- Pij2 j=1 Where PICi is the polymorphic information content of a marker i; Pij is the frequency of the jth pattern for marker i and the summation extends over n patterns

Polymorphic Information Content PIC)

n PICi = 1- Pij2 j=1 Example: Marker A has two alleles, first allele has a frequency of 30%, the second allele has a frequency of 70% PICa = 1- (0.32 + 0.72) = 1- (0.09 + 0.49) = 0.42

Polymorphic Information Content PIC)

n PICi = 1- Pij2 j=1 Example: Marker B has two alleles, first allele has a frequency of 50%, the second allele has a frequency of 50% PICb = 1- (0.52 + 0.52) = 1- (0.25 + 0.25) = 0.5

Polymorphic Information Content PIC)

n PICi = 1- Pij2 j=1 Example: Marker C has two alleles, first allele has a frequency of 90%, the second allele has a frequency of 10% PICc = 1- (0.92 + 0.12) = 1- (0.81 + 0.01) = 0.18

Polymorphic Information Content PIC)

n PICi = 1- Pij2 j=1 Example: Marker D has 10 alleles, each allele has a frequency of 10% PICd = 1- [10 x 0.12] = 1- 0.1 = 0.9

Allele frequency and Forensics

Say, we have 10 marker loci We have done adequate population genetics to know each one have a 10% distribution Test of each locus can define certain level of confidence as to what the probability is to obtain the results you are obtaining.

Allele frequency and Forensics

Locus 1, positive You are included, but every one out of 10 people has the chance to be positive locus 2, positive You are included, but every one out of 100 people has the chance to be positive at both locus 1 and locus 2 Locus 10, also posive ...