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Chapter 6: PCR methods for analysis of DNA polymorphisms

PCR-RFLP STR (microsatellites)RAPDAFLP

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Some methods to detect DNA polymorphisms

• SNP analysis: by chip analysis or PCR

• RFLP: e.g. sickle cell globin, sometimes no hybridisation necessary e.g. mitochondrial DNA

• PCR e.g. followed by sequence analysis

• PCR-RFLP: PCR, followed by restriction digest (e.g. ITS of rDNA or chloroplast DNA)

• STR: short tandem repeats or microsatelites, usually detected by PCR

• RAPD: random PCR

• AFLP: PCR of selected restriction fragments

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PCR-RFLP

• A1 & A2 are 2 alleles of one locus

• PCR primer pair is choosen to amplify this region

• Restriction digest

• DNA polymorphism results in differential cutting

• Analysis on agarose gelVery simple and cheapLimited to certain DNA

regions (ITS, cp genes,etc.)Not very polymorphic

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Different Vasconcellea (highland papaya) species from Ecuador analysed by PCR-RFLPPCR on CpDNA-region trnK1-trnK2 then digest with AfaI

‘carv’V. goudotiana

M goud long pulchra micro mon carp carv pal M

PCR-RFLP

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M goud long pulchra micro mon carp carv pal M

Some species have the same pattern (more related?)

CpDNA-region trnK1-trnK2 digest with AfaI

Intraspecific variation

PCR-RFLP

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4 intergeneric crosses:

♀ ♂C. papaya V. parviflora

C. papaya V. goudotiana

C. papaya V. cundinamarcensis

C. papaya V. quercifolia

C. papaya

F1

V. parviflora

PCR-RFLP

PCR-RFLP to analyse inheritance of organel DNA: maternal or paternal or biparental?

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M pap 1 2 3 4 5 cun

M pap 1 2 3 4 5 cun

Chloroplast DNAK1K2/AfaI

Mitochondrial DNAnad4/1-2

C. papaya (seed parent = ♀) V. cundinamarcensis (pollendonor = ♂)

⇒cpDNA als mtDNA: maternal ⇒for all intergeneric crosses tested⇒

PCR-RFLP

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VNTR=variable number of tandem repeats

• VNTRs are hypervariable due to mistakes during replication or recombination

• Microsatellites or STR (Short Tandem Repeat) or SSR (Simple Sequence Repeat): are repeats of a motif of 2, 3 or 4 nucleotides many times (such as CAGCAGCAGCAGCAGCAGCAG)

• Minisatelites were the first DNA fingerprints used in humans, they have larger motifs (tens of nucleotides) and are usually analysed by RFLP

• These types of repeats have been found in all eukaryotes studied

• Because of the high variability, they can be used to make individual DNA fingerprints

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Minisatelites are usually analysed via hybridisation = variant of RFLP-analysis see RFLP, not much used anymore

VNTR=variable number of tandem repeats

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A diploid organism has 2 alleles of a minisatellite locus, but many alleles can be present in a population

VNTR=variable number of tandem repeats

Who did it?

genetic fingerprints

If enough loci are analysed, the banding pattern is specific for

the individual

Who is the father?

M C F1 F2

Every child (C) gets half of its DNA from the mother (M) and the other half from its

father

F1 of F2?

F2

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STR= short tandem repeats

Microsatellites (STR, short tandem repeats or SSR simple sequence repeats) are usually studied by PCR, with analysis on polyacrylamide gel, detection can be by silver staining or incorporation of radioactivity or fluorescence

simple, fast, very polymorphic, much information if on several loci

development time-consuming, loci need to be searched for and sequenced

genome1 genome2

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Example:

Combination of several single locus analyses (code of primer pairs on top)S1, S2, S3: the suspectsU1 tot U7: sperm samples collected from several women that had been raped.

STR= short tandem repeats

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Single locus analyses can be combined on 1 gel if fragment sizes differ sufficiently and/or by incorporating different fluorescent colours.

STR= short tandem repeats

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RAPD

• RAPD = random amplification of polymorphic DNA• Principle: one short primer (e.g. 10 nucleotides) is

used in the PCR reaction, this primer binds where it finds homology (many places), if two primers are by chance pointing to each other and not too far away, they can give a PCR product

DNA

PCR-productprimer

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• Example: RAPD patterns of different populations of Heterodera schachtii and of some other Heterodera species (21-23)

RAPD

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Main advantages of RAPD: • Simple technique (analysis on agarose gel)• No sequence information needed to do this

type of PCR

Main disadvantage:• Not very reproducible

RAPD

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AFLP

• AFLP is a technique in which differences in restriction fragments are revealed by PCR, and this not for one locus but for a larger number of loci in one reaction

• In a first step the restriction fragments are generated by using two different enzymes (a frequent tetra-cutter and a more rare hexacutter)

• Adapters are ligated to these fragments in order to have known sequences for primer design

• Selected fragments are amplified (to have between 50-150 bands on the gel) and separated by polyacrylamide gel electrophoresis (detection by autoradiography or fluorescence)

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Amplified fragment length polymorphisms

Adapter ligation

First restriction digestion

Second restriction digestion

DNA

AFLP

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Selective fragment amplification

Restriction enzyme digestion

Adapter ligation

Selective PCR amplification

AC

GC

AFLP primer 1

Selective bases

Selective bases

common sequence 1

AFLP primer 2

common sequence 2

R1 R2

AFLP

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AFLP

• Selected fragments are amplified (to have 50-150 bands on the gel) and separated by polyacrylamide gel electrophoresis (detection by autoradiography or fluorescence)

• This selection is made by using longer primers: every extra nucleotide decreases the number of fragments by 1/4, so 2 extra nucleotides on each primer will amplify 1/256

• By repeating this second amplification with other primer pairs (other selective nucleotides) a different subset of the genome is amplified.

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AFLP

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The main advantages of AFLP are• No need for known sequences in the genome• High reproducibility• Many loci are simultaneously analysed• By changing the selective nucleotides a

different part of the genome (and thus different loci) can be analysed

• Whole genome analysis is (theoretically) possible

The main disadvantage: complex procedure

AFLP

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AFLP

Example of AFLP gel from individuals sampled from a tropical tree species

About 70 samples

Ab

ou

t 30

- 4

0 b

and

s vi

sib

le

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C. papaya V. monoica V. palandensis V. parviflora V. weberbaueri ‘carv’

Primer pair EcoRIGA/MseIACAA

AFLP analysis of Vasconcellea species: detail

Species-specific marker

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1 2 3 4 1 2 3 4

1

2

3

4

AFLP PCR-RFLP STR Sequenceanalysis

1 2 3 4

Nuclear DNA

Large number of loci

Many polymorphisms

Complex

ITS, chloroplast & mitochondrial DNA

Cheap

Fast and efficient

Little information

Low polymorphism

Nuclear and chloroplast DNA

Extremely polymorphic

Codominant

Development time long

Nuclear and chloroplast DNA

Lots of information

Expensive

Chip (SNP)analysis

Large number of loci

Many polymorphisms

Development time long

Expensive

Choice of marker

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• Depending on the level where polymorphisms need to be analysed, a different method has te be choosen

Level polymorphisms

Family > genus > species > cultivar or individual

Marker polymorphism

STR > SNP > AFLP > sequence > PCR-RFLP

• If the level of polymorphism is low, for example below species level, a highly polymorphic marker needs to be choosen such as STR

• For non-model organisms, SNP and STR are not usually available, AFLP is the fastest option, because for SNP/STR a lot of preparatory work is needed to find polymorphisms

Choice of marker

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Comparison banding patterns

PCR-RFLP AFLP RAPD

STR: for population genetics

For phylogeny: sequence alignment

For classification/diagnostics