PCR Techniques. By Ahmad Mansour Mohamed Alzohairy. Department of Genetics, Zagazig University,Zagazig, Egypt

Transcription

1 PCR Techniques By Ahmad Mansour Mohamed Alzohairy Department of Genetics, Zagazig University,Zagazig, Egypt 2005

2 PCR Techniques ISSR PCR Inter-Simple Sequence Repeats (ISSRs) By Ahmad Mansour Mohamed Alzohairy Department of Genetics, Zagazig University,Zagazig, Egypt 2005

3 What are ISSRs? A marker system called Inter-Simple Sequence Repeats (ISSRs) has only recently been developed as an anonymous, RAPDs-like approach that accesses variation in the numerous microsatellite regions dispersed throughout the various genomes (particularly the nuclear genome) and circumvents the challenge of characterizing individual loci that other molecular approaches require.

4 Microsatellites are very short (usually base-pair) stretches of DNA that are "hypervariable", expressed as different variants within populations and among different species. They are characterized by mono-, dior trinucleotide repeats, e.g., AA, or AG, CAG, that have 4-10 repeat units side-byside.

5 ISSRs, specifically target the di- and trinucleotide repeat types of microsatellite, because these are characteristic of the nuclear genome (mononucleotide types are found in the chloroplast genome and we don't want these).

6 Studies of ISSR locus heritability have demonstrated an exceedingly close approximation to classic Mendelian ratios (Tsumura et al. 1997).

7 Extremely high variability and high mapping density as compared with RFLP and RAPD data make these new dominant, microsatellite-based molecular markers ideal for producing genetic maps of individual species (Nagaoka and Ogihara 1997 (.

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9 In the past years many different protocols were tested and standardized for population studies, polymorphism estimations, strain/variety identification, Mapping etc.

10 ADVANTAGES : This method requires small quantity of DNA (5-20 ng/reaction depending up on the type of electrophoretic assay) and it is PCR based. This method provides dominant, reproducible and large number of markers. The whole analysis is automated to enable the researchers to carry out large-scale genetic mapping and population studies. This method overcomes the limitation of flanking sequence characterization as required by SSRs. LIMITATIONS: It is a dominant marker and hence not as informative as SSRs

11 Protocols List: Protocol of ISSR PCR for agarose gel electrophoresis, is a fast and efficient technique for standardizing primers and quick polymorphism testing including mapping. This method is efficient in differentiating products of 2% difference. The only drawback is the possibility of missing faint amplification products, as the sensitivity of detection is low..

12 Protocol of ISSR PCR for PAGE and silver staining, is an efficient and reliable method with a better resolving power than agarose gel. It is safer as it does not require radioactivity but is more labor intensive than agarose gels

13 Protocol of ISSR PCR using radiolabel, is the most sensitive technique among these methods. It is labour intensive and involves the risk of radioactivity handling.

14 Protocol for fluorescent ISSR (FISSR) PCR, combines both speed and sensitivity but is more expensive than other methods. This method is automated with the use of fluorescent dntps or fluorescent labeled primers and thus requires automated sequencing system. It is specially suitable for large scale genetic analyses.

15 It is important to amplify each set of samples and a particular primer twice- giving two amplification replicates. A few bands appear and disappear at random, depending on conditions and the probabilistic nature of PCR..

16 - Bands are scored as "present" for a sample and a given primer only where they occur in both replicates, and "absent" where they occur in only one replicate or neither of them. - Each fragment scored as "present" is treated as a "dominant" (amplified) band for that locus, while one scored as "absent" is treated as a "recessive" (null) band; - note that homozygous dominant and heterozygous genotypes can't be distinguished in diploid individuals.

17 This must be accommodated in statistical formulas in arriving at F-equivalent "phi" statistics

18 Some ISSR PRIMERS Name* Sequence 814 (CT)8TG 844A (CT)8AC 844B (CT)8GC 17898A (CA)6AC 17898B (CA)6GT 17899A (CA)6AG 17899B (CA)6GG HB 8 (GA)6GG HB 9 (GT)6GG HB 10 (GA)6CC HB 11 (GT)6CC HB 12 (CAC)3GC HB 13 (GAG)3GC HB 14 (CTC)3GC HB 15 (GTG)3GC

19 By Ahmed Mansour Alzohairy Department of Genetics, Zagazig University, Zagazig, Egypt