For identification of genetic variation in strains or closely related species of plants, fungi, animals and bacteria.To detect various polymorphisms in different genomic regions.Separation and analysis is to be performed on sequencer in that case fluorescently labelled primers are used during selective amplification. the bending pattern of the fragments is analysed manually or with analytical software. The separation of fragments can be done on a 6% polyacrylamide gel or automated sequencer containing pop gels. In touchdown pcr the annealing temperature is lowered by certain degrees after every PCR cycle to, improve the amplification efficiency of AFLP. Few cycles of Touchdown PCR is set to perform amplification.In selective amplification more stringent primers (primer contain additional 3 nucleotides) are used to reduce the number of fragments amplified. PCR of 20 cycles is set using the PCR product of pre-selective amplification step.Ī second round of amplification is known as a selective amplification. It is the first round of amplification, in which few fragments are selectively amplified. The second phase is the selective amplification The first phase is known as pre-selective amplification and These adapters of known sequences serves as the target for PCR amplification. Two different adapters are used one each for Eco R1 and another for Mse1. Ligation of Oligonucleotide Adapters Adapters are double-stranded short oligonucleotide sequences of usually 14 to 20 base pairs. A frequent cutter(Mse-1) identifies and cleaves 4 base pair restriction site.Ģ). A rear cutter(Eco R1) identifies 6 base pair restriction site and cleaves it A rear cutter such as Eco R1 and a frequent cutter such as Mse-1 is used producing sticky ends. The next step is the digestion with restriction endonucleuses. DNA extraction and Restriction DigestionĪFLP technique requires very good quality of intake and pure genomic DNA, which should be free from protein and contaminants. The variation in the length of fragments is analysed, which gives the estimate about genetic relationships among the individuals or the variations. The amplified products are visualised on high resolution polyacrylamide gels or automated sequencers. This marker type was named as AFLP or Amplified Fragment Length Polymorphism.ĪFLP involves the digestion of genomic DNA using restriction endonucleuses, followed by adapter ligation and PCR amplification. developed a new marker type which is the combination of both the restriction based as well as the PCR based method. PCR-based markers include SSRs, RAPDs, ILPs etc. The Example of restriction based technique is RFLP marker, In this technique the DNA is restricted with specific Restriction Endonucleases. All the molecular marker techniques fall under two categories. Markers are the tools that are used to distinguish DNA, individuals, populations or species.
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