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SCoT and RAPD
- 2. Presented by Atika Agarwal M.Sc Biotechnology IV Semester
- 3. A Genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species. It can be described as a variation that can be observed A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair or a long one, like Minisatellites.
- 5. Biochemical markers Monoterpenes Allozymes (Isozyme) DNA Based Markers Anonymous Markers Defined or polymorphic Markers PCR Based Markers Sequence Tagged Markers Hybridization Based Markers SCoT RAPD EST AFLP SCAR RFLP Minisatellite Microsatellite
- 6. RAPD & SCoT
- 7. Random Amplified Polymorphic DNA (RAPD) It is a PCR based technology. In 1990 Welsh and McClelland and Williams et al. developed this technique. In RAPD the Decamer primers will or will not amplify a segment of DNA depending on the positions that are complimentary to the primer sequence. If the priming sites are in the amplifiable region a discrete DNA product is formed through cyclic amplification.
- 8. Cell /Tissue of an individual Double stranded genomic DNA Single stranded DNA Primers annealed to template DNA Complementary strand is synthesized Isolate DNA Taq polymerases ,primer, dNTPs Denature DNA at 94 °C for 3 minute keep in tubes of PCR thermocycler Annealing of primers ( 36°C for 45 Sec) DNA is synthesized at 72°C for 2 min Amplified products were analyzed using Gel Electrophoresis 35 – 45 cycles
- 9. Template DNA Primers point in the same direction, so amplification won’t happen
- 10. Template DNA Primers too far apart so amplification won’t happen > 2,000 bases
- 11. Template DNA Primers are just the right distance apart, so fragment is amplified 100 - 1,500 bases
- 12. Amplification was performed in the following conditions: Initial Denaturation 94 ºC For 3 Min Denaturation 94 ºC For 45 Sec Annealing 36 ºC For 45 Sec 45 Cycles Extension 72 ºC For 2 Min Final Extension 72 ºC For 7 Min Composition of PCR Mix: Taq buffer A 5µL MgCl2 5µL dNTP’s 2µl Primer 2µl Taq polymerase 0.1µl Distilled water 25.9µl Template DNA 10µl Preparation Of PCR Mixture And General Methodolgy
- 13. All amplified products were resolved on 1.5% high resolution agarose gel made in 0.5X TBE buffer for 3.5 hours at 70Volt and stained with ethidium bromide (10mg/mL). The banding pattern images were acquired under UV light using a Gel Image System or Gel Imager, also known as a Gel Documentation System(Bio- Rad). ELECTROPHORESIS
- 15. ADVANTAGES It does not require blotting or hybridization Low quantities of template DNA required. In expensive No species specific probes are required for different species Quick and easy to assay Do not require any specific knowledge of the target Crude DNA preparation may be used for analysis of whole genome
- 16. 1 • Low reproducibility 2 • PCR cycling conditions greatly influence the out come 3 • Highly sensitive and complicated procedure 4 • Mismatches between primer and template may result in total absence of PCR product 5 • RAPD markers are dominant so it is cause problems whether the DNA segment is amplified from locus that are heterozygous and homozygous.
- 18. In Gene mapping DNA amplification finger printing Study of closely related species RAPD Markers help to determine specific genes in chromosomes. RAPD can be used for identification of somatic Hybrid among the developing regenerates. APPLICATIONS
- 19. SCoT
- 20. Start Codon Targeted Polymorphism (SCoT) PCR based technique developed by Collard and Mackill in 2009. Simple and novel DNA marker technique, uses 18-mer single primer in PCR and an annealing of 50 ºC. PCR products are resolved using standard agarose gel electrophoresis. SCoT based on the short conserved region flanking the start codon (ATG) in plant genes. This technique has been demonstrated high polymorphic and efficient, and successfully utilized in rice and peanut for cultivar identification and genetic diversity analysis
- 21. Amplification
- 22. Each reaction contains: 25ng Template DNA 1µl Water 6.35µl 10X PCR buffer 1.2µl Primer 1.0µl dNTPs 0.3µl Taq DNA polymerase 0.150 µl Amplification was performed in the following conditions: Initial Denaturation 94 ºC For 5 Min Denaturation 94 ºC For 1 Min Annealing 50 ºC For 1 Min 35 Cycles Extension 72 ºC For 2 Min Final Extension 72 ºC For 5 Min Preparation of PCR Mixture and General Methodolgy
- 23. All amplified products were resolved on 1.5% high resolution agarose gel made in 0.5X TBE buffer for 3.5 hours at 70Volt and stained with ethidium bromide (10mg/mL). The banding pattern images were acquired under UV light using a Gel Image System or Gel Imager, also known as a Gel Documentation System(Bio-Rad). Electrophoresis
- 25. 1 • Technically simple 2 • SCoT employs longer primers (18-mer) producing high polymorphism which is reproducible 3 • It is a dominant marker system, requires no prior sequence information and the polymorphism is correlated to functional genes and their corresponding traits 4 • The recombination levels between SCoT marker and the gene/trait are generally lower when compared with random markers such as RAPDs, ISSRs or SSRs 5 • Thus it could be used directly in marker-assisted breeding programmes. 6 • Point mutations affecting the primer binding region generate polymorphism with SCoT primers
- 27. It is reproducible, reliable, efficient and easy to use. It has been used to evaluate genetic relationships in plants. It is useful for plant breeding. It is useful for accessing genetic relationships. This marker system requires no prior knowledge about the sequence under study. It is useful for QTL mapping.
- 28. SCoT marker has emerged as a superior system when compared to RAPD. SCoT is able to give high reproducibility when compared to RAPD. Also, the annealing temperature of SCoT is higher than that of RAPD. This can be attributed to longer 18-mer primers of SCoT compared to 10-mer primers of RAPD. In some cultivars SCoT has emerged superior to ISSR when used for accessing genetic relationships. The information generated by Start Codon Targeted Polymorphism is valuable. Since it is a gene targeted marker system it produces highly specific and reliable data.