Marking the Genome-microsatellites

7/31/2019 Marking the Genome-microsatellites

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Microsatellites



What is microsatellite

• Simple Sequence Repeats (SSR)

• 1-6 bp long



Classification of Microsatellites

• Simple microsatelltes

• Composite microsatellites



Simplemicrosatellites

containonly

onekindof

repeat

sequences:

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



Compositemicrosatellites

containmore

thanonetype

repeats



Molecular Basis of Microsatellite

Polymorphism

Different by 3 repeats

• Slippage of DNA polymerase is believed to be the major cause ofmicrosatellite 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 speciesstudied to date have miocrosatellites

• In well studied mammal species, onemicrosatellite 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



2

36

1

Small Locus sizes adapt them for PCR

PCR



Microsatellites are co-dominant

markers

AD BC

Allele A

Allele B

Allele C

Allele D

BD CD AC AB BD AC BD AB

AB CD BC CC



Mendelian Inheritance of Microsatellites

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

Microsatellites are inherited as codominant markers accordingto Mendelian laws



Advantages of Microsatellite Markers

AbundantEvenly

distributedHighly

polymorphic

Smallloci

Co-dominant



Development of

microsatellite markers



Need

• SSR containing clones

• Sequences of the flanking regions of SSR



Genomic DNA

Microsatellites-enriched

Small-insert DNA Libraries (I)

Digest with several 4-bp blunt endersGel fraction of 300-600 bp

Ligation to a phagemid vector

insert

Small insert

3.4 kb

insert

Small insert

3.4 kb

insert

Small insert

3.4 kb

insert

Small insert

3.4 kb

insert

Small insert

3.4 kb

insert

Small insert

3.4 kb

insert

Small insert

3.4 kb

insert

Small insert

3.4 kb

insert

Small insert

3.4 kb

micro

Small insert3.5 kb



insert

Small insert plasmids3.5 kb

insert


insert


in

micro

sert


Conversion into single-stranded

phagemids using helper phage

Single-stranded phagemids

3.5 kb


3.5 kb


3.5 kb

micro


3.5 kb

Won’t be converted to ds

will be degraded in WT host

Using dut/ung-

CJ236 strain

u

uu

u

uu

uu

uu

u

uu

Microsatellites-enriched Libraries (II)



micro


3.5 kb

Convert into ds

using (CA)15 (e.g.)

micro

3.5 kb

micro

ds plasmids

3.5 kb

u

u

u

Transform intoWT E. coli

micro

ds plasmids

3.5 kb

Microsatellite-enriched Libraries (III)

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



Microsatellites-enriched

Libraries

CAGA

TACG

CTGT

CAACATCAG

CACCGGCGTCGCCGA

...

4 bp 5 bp



Characterizationof Microsatellites

• Isolate plasmid DNA;

• sequence clones;

• Identify clones with enough sequencesfor 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 Polymorphicinformation content

• Measures the usefulness of a marker• Informativeness in specific families



1. AA x AA

4. AA x AB

Not polymorphic

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

6. AØ x AB

2. AA x BB

5. AA x BØ

No segregation

A not segregateB segregates 1:1

A segregates 3:1,B segregates 1:1

3. AØ x ØØ Only 1 allelesegregating 1:1

7. AB x AB A segregates 3:1,B segregates 3:1

Microsatellite Genotyping



Microsatellite Genotyping

8. AØ x BØ

9. AB x ØØ

10. AA x BC

11. AØ x BC

12. AB x AC

13. AB x CD

A segregates 1:1,B segregates 1:1

A segregates 1:1, B segregates1:1, A & B alternating

2 of the 3 allelessegregating 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 alleleexisting for some individuals

All 4 allelessegregating 1:1



• PIC refers to the value of a marker for detectingpolymorphism within a population

• PIC depends on the number of detectable allelesand the distribution of their frequency.

• Bostein et al. (1980) Am. J. Hum Genet. 32:314-331.

• Anderson et al. (1993). Genome 36: 181-186.

Polymorphic Information Content PIC)



n

PICi = 1- Pij2

j=1

Where PICi is the polymorphic information contentof a marker i; Pij is the frequency

of the jth pattern for marker i and the summationextends over n patterns

Polymorphic Information Content (PIC)



n

PICi = 1- Pij2

j=1

Example: Marker A has two alleles, first allele has afrequency of 30%, the second allele has a

frequency of 70%PICa = 1- (0.32 + 0.72) = 1- (0.09 + 0.49) = 0.42




n

PICi = 1- Pij2

j=1

Example: Marker B has two alleles, first allele has afrequency of 50%, the second allele has a

frequency of 50%PICb = 1- (0.52 + 0.52) = 1- (0.25 + 0.25) = 0.5




n

PICi = 1- Pij2

j=1

Example: Marker C has two alleles, first allele has afrequency of 90%, the second allele has a

frequency of 10%PICc = 1- (0.92 + 0.12) = 1- (0.81 + 0.01) = 0.18




n

PICi = 1- Pij2

j=1

Example: Marker D has 10 alleles, each allele has afrequency 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 toknow each one have a 10% distribution

• Test of each locus can define certain level ofconfidence as to what the probability is to obtainthe results you are obtaining.



Allele frequency and Forensics

• Locus 1, positive

• You are included, but every one out of 10 peoplehas the chance to be positive

• locus 2, positive

• You are included, but every one out of 100people has the chance to be positive at bothlocus 1 and locus 2

• …

• Locus 10, also posive

• ...

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Marking the Genome-microsatellites