Transcript Slide 1

DNA polymorphisms
• Insertion-deletion length polymorphism – INDEL
• Single nucleotide polymorphism – SNP
• Simple sequence repeat length polymorphism –
mini- and micro-satellites
Restriction Fragment Length
Polymorphism (RFLP)
• RFLPs (Botstein et al. 1980) are differences in restriction
fragment lengths caused by a SNP or INDEL that create
or abolish restriction endonuclease recognition sites.
• RFLP assays are based on hybridization of a labeled DNA
probe to a Southern blot (Southern 1975) of DNA digested
with a restriction endonuclease
Labeled
Probe
Target
3’ TGGCTAGCT 5’
1
3’ TGGCTAGCT 5’
|||||||||
5’-CCTAACCGATCGACTGAC-3’
2
5’-GGATTGGCTAGCTGACTG-3’
RFLP
A C A T T GCGAA T T C A T GT A CGC A T
T GT AA CGC T T AAGT A CA T GCGT A
RFLPs
A C A T T GCGAAG T C A T GT A CGC A T
T GT AA CGC T T C AGT A CA T GCGT A
Allele A
Allele a
A
a
a
A
a
a
A
a
Ind 1 Ind 2 Ind 3 Ind 4 Ind 5 Ind 6 Ind 7 Ind 8
A C A T T GCGAA T T C A T GT A CGC A T
T GT AA CGC T T AAGT A CA T GCGT A
RFLPs
A C A T T GCGAAG T C A T GT A CGC A T
T GT AA CGC T T C AGT A CA T GCGT A
Allele A
Allele a
A
a
a
A
a
a
A
a
Ind 1 Ind 2 Ind 3 Ind 4 Ind 5 Ind 6 Ind 7 Ind 8
RFLPs
Allele B
Insertion
Allele b
B
b
b
B
b
b
B
b
Ind 1 Ind 2 Ind 3 Ind 4 Ind 5 Ind 6 Ind 7 Ind 8
Features of RFLPs
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Co-dominant
Locus-specific
Genes can be mapped directly
Supply of probes and markers is unlimited
Highly reproducible
Requires no special instrumentation
Amplified Fragment Length
Polymorphism (AFLP)
• AFLPs (Vos et al. 1995) are differences in
restriction fragment lengths caused by SNPs or
INDELs that create or abolish restriction
endonuclease recognition sites.
• AFLP assays are performed by selectively
amplifying a pool of restriction fragments using
PCR.
EcoRI
MseI
Digestion with 2
restriction
enzymes
Restriction site
adapter ligation
Selective preamplification
3’
5’
T
A
T
5’
3’
A
Amplification
3’
5’
CTT
5’
ATG
3’
Amplified Fragment Length
Polymorphism (AFLP)
Polymorphisms betwwen genotypes may arise
from:
– Sequence variation in one or both restriction sites
– Sequence variation in the region immediately
adjacent to the restriction sites
– Insertions or deletions within an amplified fragment
AFLP
The predicted number of DNA
fragments amplified by AFLP primers
with n selective nucleotides is
N is genome size in base-pairs
b is the number of nucleotides in the
recognition site of a restriction
endonuclease
AFLP
• DNA restriction fragments produced by a six-base cutter
(n = 6) in soybean (N = 1 X 109 bp, ~1,000 Mb/1C)
N = genome size in
base-pairs
b = the No. of
nucleotides in in the
recognition site
n = No. of selective
nucleotides
n
Calculation
No. of restriction fragments
0
1/46 (1 X 109)
244,141
1
1/46 (1 X 109) 1/42
15,259
2
1/46 (1 X 109) 1/44
954
3
1/46 (1 X 109) 1/46
60
Features of AFLPs
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Very high multiplex ratio
Very high throughput
Off-the-shelf technology
Fairly reproducible
Dominant and co-dominant
Simple Sequence Repeats (SSR)
• SSRs or microsatellites (Nakamura et al. 1987)
are tandemly repeated mono-, di-, tri-, tetra-,
penta-, and hexa-nucleotide motifs
• SSR length polymorphisms are caused by
differences in the number of repeats
• Assayed by PCR amplification using pairs of
oligonucleotide primers specific to unique
sequences flanking the SSR
Individual 1 (AC)x9
51 bp
Chloroplast SSRs of pine
Powell et al. 1995. Proc Natl Acad
Sci U S A. 92(17): 7759–7763.
SSR
Individual 2 (AC)x11
55 bp
Features of SSRs
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Highly polymorphic
Highly abundant and randomly dispersed
Co-dominant
Locus-specific
High throughput
Can be automated
SSR
• Sources
• SSRs are often found in cDNA and genomic
DNA sequences
• SSRs are developed by screening genomic
DNA libraries enriched for one or more repeat
motifs.
• SSR-enriched libaries can be commercially
purchased
SSR
Repeat Motifs
• AC repeats tend to be more abundant than other di-nucleotide
repeat motifs in animals (Beckmann and Weber 1992)
• The most abundant di-nucleotide repeat motifs in plants, in
descending order, are AT, AG, and AC (Lagercrantz et al. 1993;
Morgante and Oliveri 1993)
• Typically, SSRs are developed for di-, tri-, and tetra-nucleotide
repeat motifs
• CA and GA have been widely used in plants
• Tetra-nucleotide repeats have the potential to be very highly
polymorphic; however, many are difficult to amplify
Cleaved Amplified Polymorphic
Sequences (CAPS)
• CAPS polymorphisms are differences in restriction
fragment length caused by SNPs or INDELs that create
or abolish restriction endonuclease recognition sites in
PCR amplicons produced by locus-specific
oligonucleotide primers (Tragoonrung et al. 1992;
Konieczny and Ausubel 1993)
• Assays are performed by digesting locus-specific PCR
amplicons with one or more restriction enzymes and
separating the digested DNA on gels
CAPS
Forward Primer
Indiv.
1
Indiv.
2
Reverse Primer
Forward Primer
Reverse Primer
EcoRI
EcoRI
Indiv.
1
Indiv.
2
EcoRI
Features of CAPS
• Locus-specific - PCR-based assay for a marker
with known DNA sequence
• Method to map markers without Southern
blotting
• Co-dominant and dominant
Random Amplified Polymorphic DNA (RAPD)
• DNA polymorphism produced by
rearrangements at or between oligonucleotide
primer binding sites in the genome (Williams et
al. 1990)
• Assays are performed using single short
oligonucleotide primers of arbitrary sequence
(typically 10-mers)
RAPDs
3’
5’
Indiv. 1
5’
3’
Indiv. 2
3’
5’
5’
3’
(Brahm et al. 2000)
Features of RAPDs
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Simple assay
Dominant
Unrestricted access to primers
Requires little initial investment
Not very reproducible