RTPCR RAPD PCR and AFLP PCR with 3D animation

RT-PCR (Reverse transcriptase PCR)
This type of PCR use mRNA or RNA as an initial sample. mRNA or RNA firstly convert it to cDNA by reverse transcriptase enzyme and then amplification of this cDNA takes place.
Isolation of mRNA from cell is done by affinity chromatography through the deoxy thymidine ligand. After that conversion of mRNA into cDNA occurs in first strand reaction and cDNA amplification occur in second strand reaction. RT-PCR (Reverse transcriptase PCR)This whole process is carried out in one tube, for first reaction, deoxy thymidine act as primer for reverse transcriptase and formation of ss cDNA takes place as reverse transcriptase synthesize the cDNA and simultaneously degrade the mRNA which act as template for cDNA synthesis. RT-PCR (Reverse transcriptase PCR)After that this cDNA is converted into ds cDNA with the help of reverse transcriptase enzyme by using ss cDNA act as template and the non hybridized ss cDNA present is between ds cDNA is cleaved by S1 endonuclease.RT-PCR (Reverse transcriptase PCR) This reaction is carried out at 37°C with the help of reverse transcriptase. RT-PCR (Reverse transcriptase PCR)The reverse transcriptase use in both reactions. name of some other reverse transcriptase, AMV (Avian Myeloblastosis Virus) reverse transcriptase, Mo-MLV (Moloney Murine Leukemia Virus) reverse transcriptase.RT-PCR (Reverse transcriptase PCR)
Now second reaction takes place in which deoxy thymidine act as one primer and we need a second primer which is specific for cDNA and made by 5’UTR of mRNA, because it is complementary to 5’UTR of mRNA. ~35 cycle are required to carry out amplification of cDNA. cDNA is used to form cDNA libraries and diagnosis of genetic diseases. Random amplified polymorphic DNA (RAPD) or AP-PCR (Arbitrary primed PCR)RT-PCR (Reverse transcriptase PCR)RT-PCR (Reverse transcriptase PCR)
Random amplified polymorphic DNA (RAPD) or AP-PCR (Arbitrary primed PCR)
It is another technique based on PCR. It is use to compare PCR amplification profile of different sample collected from same place or different place in one PCR reaction. This is specially use in forensic science to amplify the different sample received from crime place in one PCR reaction. This technique in turn detect polymorphism among different sequence This task is accomplished because in this PCR arbitrary primer of 8-12 nucleotide are use, but mostly decamer is use and DNA synthesis carried out by Taq DNA polymerase.Random amplified polymorphic DNA (RAPD) or AP-PCR (Arbitrary primed PCR). Random amplified polymorphic DNA (RAPD) or AP-PCR (Arbitrary primed PCR)
In this technique large target DNA sequences are used which may possess two or more binding site for arbitrary primer and if primer hybridize the place which allow the target strands amplification from both place then it get amplify from both place thus polymorphism can be shown between different sequence because both primer had different binding site one each strand. Thus it act as dominant genetic marker and use to identify the polymorphism in the phylogeny among diverse animal and plant species.Random amplified polymorphic DNA (RAPD) or AP-PCR (Arbitrary primed PCR)
Steps like denaturation , annealing and extension in RAPD are same as in standard PCR but here cycle repetition occurs 40 -50 times. In RAPD more than 128 primers are used. Results are seen on gel after the completion of reaction. Random amplified polymorphic DNA (RAPD) or AP-PCR (Arbitrary primed PCR)EtBr is used to visualize the bands on gel. Here previous knowledge of target DNA sequence is not required. RAPD in not able to differentiate the heterozygousity or homozygousity between amplified DNA sequences. Random amplified polymorphic DNA (RAPD) or AP-PCR (Arbitrary primed PCR)

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