14. marking the genome-microsatellites - auburn university...• pic refers to the value of a marker...
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Microsatellites
What is microsatellite
• Simple Sequence Repeats (SSR)• 1-6 bp long
Classification of Microsatellites
• Simple microsatelltes• Composite microsatellites
Simplemicrosatellites
contain only one kind of
repeat sequences:
(GT)n (AC)n (AG)n
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 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 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
2
361
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 according to Mendelian laws
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
Genomic DNA
Microsatellites-enriched Small-insert DNA Libraries (I)
Digest with several 4-bp blunt enders
Gel fraction of 300-600 bp
Ligation to a phagemid vector
insert
Small insert3.4 kb
insert
Small insert3.4 kb
insert
Small insert3.4 kb
insert
Small insert3.4 kb
insert
Small insert3.4 kb
insert
Small insert3.4 kb
insert
Small insert3.4 kb
insert
Small insert3.4 kb
insert
Small insert3.4 kb
micro
Small insert3.5 kb
insert
Small insert plasmids3.5 kb
insert
Small insert plasmids3.5 kb
insert
Small insert plasmids3.5 kb
in
micro
sert
Small insert plasmids3.5 kb
Conversion into single-stranded phagemids using helper phage
Single-stranded phagemids3.5 kb
Single-stranded phagemids3.5 kb
Single-stranded phagemids3.5 kb
micro
Single-stranded phagemids3.5 kb
Won’t be converted to dswill be degraded in WT host
Using dut/ung-CJ236 strain
u
uu
u
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Microsatellites-enriched Libraries (II)
micro
Single-stranded phagemids3.5 kb
Convert into ds
using (CA)15 (e.g.)
micro
3.5 kb
micro
ds plasmids
3.5 kb
u
u
u
Transform into WT E. coli
micro
ds plasmids3.5 kb
Microsatellite-enriched Libraries (III)
According to Ostrander et al., 1992: PNAS 89:3419
Microsatellites-enriched Libraries
CAGATACGCTGT
CAACATCAGCACCGGCGTCGCCGA
...
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
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 allele segregating 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 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
• 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. 32:314-331.
• Anderson et al. (1993). Genome 36: 181-186.
Polymorphic Information Content PIC)
nPICi = 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)
nPICi = 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)
nPICi = 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)
nPICi = 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)
nPICi = 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
Polymorphic Information Content PIC)
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• ...
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