Rflp,rapd&aflp
- 1. ANEESHA – A VI SEMESTER BSc.BOTANY
- 3. • A molecular marker is a molecule containing within a sample taken from an organism or other matter
- 4. • A genetic marker is gene or DNA sequence with a known location on a chromosome that cam be used to identify individual or species • It may be a short DNA sequence
- 5. Should be easy, fast and cheap to detect Should be reproducible Should be polymorphic Should have co-dominant inheritance to allow discrimination between homo and heterozygote in diploids
- 6. RFLP - Restriction Fragment Length Polymorphism AFLP - Amplified Fragment Length Polymorphism RAPD - Random Amplification of Polymorphic DNA SSLP - Simple Sequence Length Polymorphism
- 7. VNTR - Variable Number Tandem Repeats SSR - Simple Sequence Repeats SNP - Single Nucleotide Polymorphism STR - Short Tandem Repeats RAD - Restriction site Associated DNA
- 8. RFLP • Termed by Botstein-1980 • Combination of a probe and restriction enzyme (RE) to identify polymorphic DNA sequence using Southern blotting
- 9. STEPS 1. DNA is isolated 2. DNA is digested with RE 3. Electrophoresis 4. Blotted on a membrane and probed with a labeled clone 5. The fragments to which the probe has hybridized are detected by autoradiography
- 11. DISADVANTAGE ● Highly pure DNA ● It is time consuming ● Laborious and expensive
- 12. RAPD • PCR based molecular technique • It is used to analyze the genetic diversity of an individual by using random primers
- 13. STEPS 1. An arbitrary oligonucleotide primer, usually 9 to 10 bp long is added to a sample of plant chromosomal DNA 2. It will pair with the chromosomal DNA at many sites, sometimes including opposite strands on the target DNA 3. DNA region will be amplified via PCR 4. Products are by visualized following polyacrylamide gel electrophoresis 5. Analysis of data
- 15. ADVANTAGES The same (universal) set of oligonucleotide primers can be used for all plant species No genomic libraries, radioactivity, Southern transfers, or DNA hybridization reactions are required, so a large number of samples may be easily and rapidly characterized The process can be automated
- 17. • It involves PCR amplification of genomic restriction fragments generated by specific RE and oligonucleotide adaptors of few nucleotide bases
- 18. STEPS 1. DNA fragments obtained from digestion with restriction enzymes 2. Ligation of oligonucleotide adapters to the digestion products 3. Selective amplification by the PCR 4. Gel electrophoresis 5. Analysis of data
- 20. RAPD RFLP 1.Quantity of DNA required for analysis is small(10-50 ug ) 1.Large quantity of purified DNA required i.e.2-10 ug 2.Same primers with arbitrary sequence can be used for different species 2.Different species specific probes are required 3.Fewer steps in procedure therefore it is rapid 3.Comparitively slower processing due to more steps involved 4.Technique comparatively less reliable 4.Technique comparatively reliable 5.Cannot detect allelic variants 5.Can detect allelic variants 6. 1-10 loci detected 6. 1-3 loci detected 7.Method involves a) Extraction of DNA b) Amplification by PCR using random primers c) Gel electrophoresis of amplified DNA and visualization of markers 7.Method involves a) Digestion of extracted DNA by restriction enzymes b) Gel electrophoresis c) Southern blot