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ppt AFLP n RFLP by yeni - Copy.pptx

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ppt AFLP n RFLP by yeni - Copy.pptx

  1. 1. PRINCIPLEOFR A P D RAPD is a PCR based technique for identifying genetic variation. It involves use of single arbitrary primer in a PCR reaction, resulting in amplification of many discrete DNA. RAPD technology provides a quick and efficient screen for DNA sequence based polymorphism at a very large number of loci.
  2. 2. RAPD is a method develop in 1990 similar to PCR. It is different from conventional PCR as it need one primer for amplification. The size of primer is shorter(10 nucleotides) therefore less specific. - the primers can be designed without the experimenter having any genetic information for the organism being tested. Genomic DNA normally has complimentary sequences to RAPD primers at many locations. The RAPD technology has provided a quick and efficient screen for DNA-sequence polymorphisms at a very large no of loci. - Normally, a few (3-20) loci can be amplified by one single RAPD primer.
  3. 3. ADVANTAGES OF RAPD Main advantages of the RAPD technology include (i) suitability for work on anonymousgenomes. (ii)applicability to problems where onlylimited quantities of DNA are available. (iii) efficiency and low expense
  4. 4. DISADVANTAGES OF RAPD Amplification either occurs at a locus or it does not, leading to scores based on band presence orabsence. This means that homozygotes and heterozygotes cannot be distinguished. Nothing is known about the identity of the amplification products unless the studiesare supported by pedigree analysis.
  5. 5. CONCLUSI ON RAPD is probably the easiest and cheapest methods for laboratory just beginning to use molecular markers. RAPD markers have found a wide range of applications in -gene mapping, - population genetics, -molecular evolutionarygenetics - plant and animal breeding. This is mainly due to the speed, cost and efficiency of the RAPD technique to generate large numbers of markers in a short period compared with previous methods.
  6. 6. R E S T R I C T I O N E N D O N U C L E A S E S Enzymes that cleave DNA molecules atspecific nucleotide sequences. Shorter the recognition sequence, the greaterthe number of fragments generated. Restriction enzymes are isolated from a widevariety of bacterial genera For example, HindII enzyme cuts at GTGCAC or GTTAAC.
  7. 7. A N A L Y S I STECHNIQUE fragmenting a sample of DNA by a restriction enzyme resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis. Then transferred to a membrane via the Southern blot procedure.
  8. 8. SOUTHERNBLOTTING A method to visualize specific segments ofDNA– usually a particular gene.  Uses radioactive probes that bind to the specific DNA segments.
  9. 9. STEPS • Soak gel in basic solution to separate DNA strands • Transfer DNA on to a nylon membrane (spacing of DNA is maintained) • Incubate with radioactive probe for specificsegment • Wash away unbound probe • Detect probes using x-ray film autoradiograph

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