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Page 1: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

DNA polymorphisms

• Insertion-deletion length polymorphism – INDEL• Single nucleotide polymorphism – SNP• Simple sequence repeat length polymorphism –

mini- and micro-satellites

Page 2: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

Labeled 3’ TGGCTAGCT 5’Probe 3’ TGGCTAGCT 5’ |||||||||Target 1 5’-CCTAACCGATCGACTGAC-3’ 2 5’-GGATTGGCTAGCTGACTG-3’

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

Page 3: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

RFLP

Page 4: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

Allele A

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A a aa A aa A

Ind 1 Ind 2 Ind 5Ind 3 Ind 4 Ind 8Ind 6 Ind 7

RFLPs

Page 5: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

Allele A

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Ind 1 Ind 2 Ind 5Ind 3 Ind 4 Ind 8Ind 6 Ind 7

RFLPs

Page 6: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

Allele B

Allele b

B b bb B bb B

Ind 1 Ind 2 Ind 5Ind 3 Ind 4 Ind 8Ind 6 Ind 7

Insertion

RFLPs

Page 7: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

Features of RFLPs

• Co-dominant• Locus-specific• Genes can be mapped directly• Supply of probes and markers is unlimited• Highly reproducible• Requires no special instrumentation

Page 8: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

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.

Page 9: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

Digestion with 2 restriction enzymes

Restriction site adapter ligation

EcoRI MseI

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5’3’

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Selective preamplification

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A T G

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Amplification

Page 10: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

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

Page 11: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

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

Page 12: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

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

Page 13: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

Features of AFLPs

• Very high multiplex ratio• Very high throughput• Off-the-shelf technology• Fairly reproducible • Dominant and co-dominant

Page 14: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

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

Page 15: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

Individual 1 (AC)x9 Individual 2 (AC)x11

51 bp55 bp

Powell et al. 1995. Proc Natl Acad Sci U S A. 92(17): 7759–7763.

Chloroplast SSRs of pine

SSR

Page 16: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

Features of SSRs

• Highly polymorphic• Highly abundant and randomly dispersed• Co-dominant • Locus-specific• High throughput • Can be automated

Page 17: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

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

Page 18: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

SSRRepeat 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

Page 19: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

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

Page 20: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

Indiv. 1

Indiv. 2

Forward Primer

Reverse Primer

Forward Primer

Reverse Primer

Indiv. 1

Indiv. 2

EcoRI EcoRI

EcoRI

CAPS

Page 21: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

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

Page 22: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

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)

Page 24: DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini-

Features of RAPDs

• Simple assay• Dominant• Unrestricted access to primers• Requires little initial investment• Not very reproducible


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