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15 molecular markers techniques


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markers assisted breeding methods

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15 molecular markers techniques

  1. 1. Molecular Markers DNA markers Techniques
  2. 2. DNA markers Restriction fragment length polymorphism (RFLP) Randomly amplified polymorphic DNA (RAPD) Amplified fragment length polymorphism (AFLP) SSR SNP
  3. 3. RFLP Botstein et al. (1980) first used DNA restriction fragment length polymorphism (RFLP) To recognize: Neutral variation at the DNA level SNPs within a gene or between genes or Variable number of tandem repeats present between genes Out come: Accelerated the construction of molecular linkage maps Improved 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.
  4. 4. 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 2000bp 2. cDNA cloning 3. PCR amplification
  5. 5. Limitations: Requires large amounts of high quality DNA Low genotyping throughput Difficult to automate Involves radioactive methods so its use is limited to specific laboratories RFLP 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 RFLP 1. co-dominant 2. reproducible 3. simple methodology 4. 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)
  6. 6. Molecular Basis of RFLP
  7. 7. 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 loci visible in conventional agarose gel electrophoresis as the presence or absence of a particular RAPD band RAPDs predominantly provide dominant markers
  8. 8. 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 buffers deoxynucleotide triphosphates (dNTPs) Mg2+ concentration cycling parameters source of Taq polymerase condition and concentration of template DNA primer concentration
  9. 9. 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 detected small DNA samples (1–100 ng) only required relatively reproducible across laboratories
  10. 10. 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 specificities and combinations otherwise the assays can be carried out using off-the-shelf technology. (vii) There are relatively high technical demands in AFLP analysis including radio-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)
  11. 11. 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
  12. 12. 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.
  13. 13. SSR Variants 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)
  14. 14. Molecular Basis of SSR
  15. 15. SSRs are characterized by: Hypervariability Reproducibility Codominant nature Locus specificity random dispersion throughout most genomes More variable than RFLPs or RAPDs. The advantages: Readily analysed by PCR and easily detected on PAGE SSLPs with large size differences - detected on agarose gels SSR markers can be multiplexed (by pooling independent PCR products or by true multiplex-PCR) genotyping throughput is high and can be automated start-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 disadvantages Labour intensive development process particularly when screening from G DNA High start-up costs for automated methods.
  16. 16. SNP Pronounced 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 SNPs 100 000 of which result in an RFLP C/T transitions constitute 67% of the SNPs in humans Similar is the case with plants (Edwards et al., 2007a) 2/3 of SNPs involve the replacement of C / T transitions Single 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 SNP 90% of all human genetic variation are SNPs and occur every 100–300 bases
  17. 17. Barley Soybean Sugarbeet Maize Cassava Potato 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 genes gene splicing, transcription factor binding the sequence of non-coding RNA the intergenic regions between genes at different frequencies in different chromosome regions
  18. 18. 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.
  19. 19. Approaches adopted for discovery of novel SNPs: vitro discovery, where new sequence data is generated silico methods that rely on the analysis of available sequence data III.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 hybridization Primer extension Oligonucleotide ligation Invasive cleavage Chagné et al. (2007) added three methods to this list Sequencing Allele-specific PCR amplification and DNA conformation method Enzymatic cleavage method - Approaches for discovery of SNPs
  20. 20. 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 assay b) Molecular beacon These can be used in high-density oligonucleotide chips Approaches for discovery of SNPs
  22. 22. 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 query c) SNP and are then extended by a single ddNTP (SBE) in cycle sequencing reactions d) 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 template Both primers must have perfect base pair complementarity at the ligation site which makes it possible to discriminate two alleles at a SNP site
  23. 23. 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.
  24. 24. Oligonucleotide hybridization analysis An 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.
  25. 25. 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