Molecular Markers

DNA markers Techniques

DNA markers

Restriction fragment length polymorphism (RFLP)Randomly amplified polymorphic DNA (RAPD)Amplified fragment length polymorphism (AFLP)SSRSNP

RFLP

Botstein et al. (1980) first used DNA restriction fragment length polymorphism (RFLP)

To recognize:Neutral variation at the DNA levelSNPs within a gene or between genes orVariable number of tandem repeats present between genes

Out come:Accelerated the construction of molecular linkage mapsImproved the accuracy of gene location and Reduced the time required to establish a complete linkage map

Technique:The digestion of purified DNA using restriction enzymes leads to the formation of

RFLPs - a molecular fingerprint - unique to a particular individual

Six base specific RE - cleave the DNA at every 4096 bases on average (46)A genome of 109 bases - produce around 250,000 restriction fragments of variable

length - a continuous smear image.

RFLP workflow from DNA extraction to radio-autograph

Molecular probes are DNA fragments isolated and individualized by

1. PstI Genomic Cloning – single copy fragments - 500 to 2000bp2. cDNA cloning3. PCR amplification

Limitations:Requires large amounts of high quality DNALow genotyping throughputDifficult to automateInvolves radioactive methods so its use is limited to specific laboratoriesRFLP probes must be physically maintained and therefore difficult to share between laboratories.The level of RFLP is relatively low selection for polymorphic parental lines is a limiting step, therefore a complete map

Advantages of RFLP1. co-dominant2. reproducible 3. simple methodology4. requires no special instrumentation 5. Cleaved amplified polymorphic sequence (CAPS) marker (PCRRFLP)

- digesting a PCR-amplified RFLP fragment with one or several restriction enzymes, and detecting the polymorphism by the presence/absence restriction sites (Konieczny and Ausubel, 1993)

Molecular Basis of RFLP

RAPD

A single, random-sequence oligonucleotide primer in a low stringency PCR (35–45°C) simultaneously amplifies several discrete DNA fragments

random amplified polymorphic DNA (RAPD) by Williams et al. (1990)

arbitrary primed PCR (AP-PCR) by Welsh and McClelland (1990)

DNA amplification fingerprinting (DAF) by Caetano-Anollés et al., (1991)

10-mer oligonucleotide several discrete DNA products up to 3 kb are amplified (amplicons) these are considered to originate from different genetic locivisible in conventional agarose gel electrophoresis as the presence or absence of a particular RAPD bandRAPDs predominantly provide dominant markers

Advantages:(i)Neither DNA probes nor sequence information is required for the design of primers (ii)No blotting or hybridization steps – quick, simple and efficient technique (iii)Small amounts of DNA (about 10 ng /rxn)(iv)Can be automated(v)Capable of detecting higher levels of polymorphism than RFLP(vi)Can be applied to virtually any organism(vii) The primers universal and so,can be used for any species(viii)RAPD products of interest can be cloned, sequenced and converted into PCR-based markers like

Sequence Tagged Sites (STS) Sequenced Characterized Amplified Regions (SCAR) (Paran and Michelmore, 1993)

Limitations: Reproducibility is questionable due to factors such as PCR buffersdeoxynucleotide triphosphates (dNTPs)Mg2+ concentrationcycling parameterssource of Taq polymerasecondition and concentration of template DNAprimer concentration

AFLP (selective restriction fragment amplification - SRFA)

Amplified fragment length polymorphism (Zabeau and Voss, 1993; Vos et al., 1995)

The selective PCR amplification of restriction fragments from a gDNA double-digest of under high stringency conditions

Combination of polymorphism at RE sites and hybridization of arbitrary primers

50 to150 bp are amplified and polymorphism detectedsmall DNA samples (1–100 ng) only requiredrelatively reproducible across laboratories

LIMITATIONS TO THE AFLP ASSAY(i)The maximum polymorphic information content for any bi-allelic marker is 0.5.(ii)High quality DNA is needed(iii)Proprietary technology is needed to score heterozygotes and ++ homozygotes(iv) AFLP markers cluster densely in centromeric regions in species with large genomes, e.g. barley (Qi et al., 1998) and sunflower (Gedil et al., 2001). (v) Developing locus-specific markers from individual fragments can be difficult(vi) AFLP primer screening is often necessary to identify optimal primer specificitiesand combinations otherwise the assays can be carried out using off-the-shelftechnology. (vii) There are relatively high technical demands in AFLP analysis includingradio-labelling and skilled manpower.(viii) Marker development is complicated and not cost-effective(ix) Reproducibility is relatively low compared to RFLP and SSR markers

genome-wide Bare-1 retrotransposon-like markers in barley (Waugh et al., 1997) diploid Avena (Yu and Wise, 2000)

cDNA-AFLP technique (Bachem et al., 1996)

Application of standard AFLP protocol to a cDNA template

e.g., Transcripts with altered expression during race specific resistance reactions

For the isolation of differentially expressed genes from a specific chromosome region using aneuploids

Construction of genome wide transcription maps

Simple Sequence Length Polymorphisms (SSLP)

• SSLPs are arrays of repeat sequences that display length variations, different alleles containing different numbers of repeat units

• Unlike RFLPs that can have only two alleles, SSLPs can be multi-allelic as each SSLP can have a number of different length variants. There are two types of SSLP, both of which were described in

• Minisatellites, also known as “variable number of tandem repeats (VNTRs)”, in which the repeat unit is up to 25 bp in length;

• Microsatellites or simple tandem repeats (STRs), whose repeats are shorter, usually “dinucleotide” or “tetranucleotide” units.

SSRVariants 1. Microsatellites 2. short tandem repeats (STRs)3. sequence-tagged microsatellite sites (STMS)

A. repeat units 1–6 bp long B. Di-, tri- and tetranucleotide repeats – (CA)n, (AAT)n and (GATA)n C. widely distributed in genomes (plants &animals (Tautz and Renz, 1984).

Advantages: high level of allelic variation Flanks of SSR motifs - templates for specific primers to amplify the SSR

alleles via PCR Referred to as simple sequence length polymorphisms (SSLPs) Mutation rates of SSR are about 4 × 104–5 × 106 /allele/generation (Primmer

et al., 1996). Mutation mechanism -‘slipped strand miss pairing’ (Levinson and Gutman,

1987)

Molecular Basis of SSR

SSRs are characterized by:HypervariabilityReproducibilityCodominant natureLocus specificity random dispersion throughout most genomesMore variable than RFLPs or RAPDs.

The advantages:

Readily analysed by PCR and easily detected on PAGESSLPs with large size differences - detected on agarose gelsSSR markers can be multiplexed (by pooling independent PCR products or by true multiplex-PCR)genotyping throughput is high and can be automatedstart-up costs are low for manual assay methods (once the markers have been developed)SSR assays require only very small DNA samples(ca.100 ng / individual)

The disadvantagesLabour intensive development process particularly when screening from G DNAHigh start-up costs for automated methods.

SNPPronounced as snip - an individual nucleotide base difference There are three types recognized Transitions (C/T or G/A) Transversions (C/G, A/T, C/A or T/G)

e.g., AAGCCTA AAGCTTA The two alleles are C and T. Indels

Human genome has at least 1.42 million SNPs100 000 of which result in an RFLPC/T transitions constitute 67% of the SNPs in humansSimilar is the case with plants (Edwards et al., 2007a)2/3 of SNPs involve the replacement of C / T transitionsSingle base variants in cDNA (mRNA) are also SNPs - insertions and deletions (indels) in the genome.Nucleotide base - the smallest unit of inheritance, SNPs

- Ultimate form of molecular marker.1% of the population should have SNP90% of all human genetic variation are SNPs and occur every 100–300 bases

BarleySoybeanSugarbeetMaizeCassavaPotato

Typical SNP frequencies are in the range of one SNP every 100–300 bp

SNPs may fall within coding sequences of genes

- if same polypeptide then synonymous - if different polypeptide then non-synonymous

non-coding regions of genesgene splicing,transcription factor binding the sequence of non-coding RNA

the intergenic regions between genes at different frequencies in different chromosome regions

Of the 3–17 million SNPs found in the human genome,5% are expected to occur within genes.Therefore, each gene may be expected to contain ca.6 SNPs.

Approaches adopted for discovery of novel SNPs:

I.in vitro discovery, where new sequence data is generatedII.in silico methods that rely on the analysis of available sequence dataIII.Indirect discovery, where the base sequence of the polymorphism remains unknown

SNP genotyping methods and chemistries:

Sobrino et al. (2005) classified SNP genotyping assays into 4 groups (based on the molecular mechanisms)

Allele-specific hybridizationPrimer extensionOligonucleotide ligationInvasive cleavage

Chagné et al. (2007) added three methods to this listSequencingAllele-specific PCR amplification andDNA conformation methodEnzymatic cleavage method -

Approaches for discovery of SNPs

1. Allele-specific hybridization

Hybridization between two DNA targets differing at one nucleotide position (Wallace et al., 1979)

Two allele-specific probes labelled with a probe-specific Fluor dye and a generic Quencher that reduces fluorescence in the intact probe

5' exonuclease activity of Taq polymerase cleaves the copml probe distancing the Quencher from flour

a) TaqMan assayb) Molecular beacon

These can be used in high-density oligonucleotide chips

Approaches for discovery of SNPs

MOLECULAR BECONS

2. Primer extension: a) Mini-sequencing, single-base extension or the GOOD assay (Sauer et al., 2002)b) Employs oligonucleotides which anneal immediately upstream of the queryc) SNP and are then extended by a single ddNTP (SBE) in cycle sequencing

reactionsd) Thermo stable proof-reading DNA polymerases ensure the complementary

ddNTP is incorporated.e) ddNTP terminators that are labelled with different fluorescent dyes are used

SNaPshot (Applied Biosystem) uses differential fluorescent labelling of the four ddNTPs in a SBE reaction

SNP-IT (Orchid Biosciences)

3. Oligonucleotide ligation (OLA)ligase joins two oligonucleotides covalently when they hybridize next to one another

on a DNA templateBoth primers must have perfect base pair complementarity at the ligation site which

makes it possible to discriminate two alleles at a SNP site

SNP

The advantages of SNPs are their abundant numbers and the fact that they can be typed by methods that do not involve gel electrophoresis. This is important because gel electrophoresis has proved difficult to automate so any detection method that uses it will be relatively slow and labor-intensive. SNP detection is more rapid because it is based on oligonucleotide hybridization analysis.

Oligonucleotide hybridization analysisAn oligonucleotide is a short single-stranded DNA molecule, usually less than 50 nucleotides in length, that is synthesized in the test tube. If the conditions are just right, then an oligonucleotide will hybridize with another DNA molecule only if the oligonucleotide forms a completely base-paired structure with the second molecule. If there is a single mismatch - a single position within the oligonucleotide that does not form a base pair - then hybridization does not occur.

Oligonucleotide hybridization

Oligonucleotide hybridization can therefore discriminate between the two alleles of a SNP. Various screening strategies have been devised including “DNA chip” technology and solution hybridization techniques