Functions of Taq DNA Polymerase in PCR Reaction

Amplified Fragment Length Polymorphism (AFLP Marker) use restriction enzymes to digest genomic DNA and adaptors to ligate digested DNA fragments and pre-selective and selective PCR amplification of ligated fragments and vertical gel electrophoresis by using denature polyacrylamide gel to separate amplified fragments, DNA digest with two restriction enzymes which cut un-evenly overhang sticky ends such as., Mse-1 (Cut Frequently) and ECOR-1 (Cut Rarely), Component of restriction digestion genomic DNA in microliter: Genomic DNA and any kind of arbitrary primer (OPA) and Mse-1 and BSA (Come with Mse-1) and ECOR-1 and deionized water then vortex samples and incubate at 37 degree Celsius for 3 hours with agitate every hour after that inactivate enzymes at 70 degree Celsius for 15 minutes Next use adaptors of known sequence as priming sites to ligate sticky ends of digested DNA fragments, Ligation Mix Adaptors of AFLP Marker: ECOR-1 adaptor and Mse-1 adaptor and T4 DNA ligase 10X buffer and T4 DNA ligase units and deionized water add 10 microliters of ligation mix to 50 microliter digested DNA after that vortex samples and incubate at room temperature for 3 hours with agitate every hour, Component of ECOR-1 adaptor and Mse-1 adaptor and PCR profile for each mix reagent: One selective PCR consist of adaptor sequences of each restriction enzymes and one arbitrary nucleotide (OPA) to amplify only subset of digested DNA fragments and after finish store tubes at minus 20 degree Celsius after that perform pre-selective PCR amplification to amplify specific subsets of digested DNA fragments using combination of selective primers at 3 ends which are Adenosine nucleotide to ECOR-1 adaptor and Cytosine nucleotide to Mse-1 adaptor that complementary to adaptors and restriction sites to select only subset fragments, Component of pre-selective PCR amplification: DNA template from restriction/ligation and ECOR-1 adaptor with Adenosine nucleotide (As primer) and Mse-1 adaptor with Cytosine nucleotide (As primer) and dNTPs and 10X PCR buffer and MgCl2 and Taq DNA polymerase and deionized water, Reaction of Pre-selective PCR amplification (26 Cycles) and after finish add 100 microliter of sterile water to PCR product, After that undergo selective PCR amplification by adding two nucleotide of Adenosine and one nucleotide of Cytosine to primer at 3 ends to again select subset fragments (Be Attention) More selective nucleotide detect less polymorphism so must only add three nucleotide, Component of selective PCR amplification: Diluted DNA template from pre-selective PCR reaction and ECOR-1 with selective nucleotide (AAC) and Mse-1 with selective nucleotide (AAC) and dNTPs and 10X PCR buffer and MgCl2 and Taq DNA polymerase and deionized water, Selective PCR reaction include two phases (First phase involve 12 Cycles with decrease temperature by 0.7 degree Celsius) while (Second phase include 23 Cycles) and after finish all phases add 8 microliters Formamide loading buffer to PCR product, Polymorphism of AFLP Marker detect by vertical gel electrophoresis using denature polyacrylamide gel to visualize separated fragments by radioisotopes or fluorescent dyes or silver stain to score bands as presence (1) or absence (0), AFLP Marker allow quick scan of whole genome because large number of generated bands give highly informative fingerprint and it doesn't require prior sequence information or probe therefore AFLP Marker classify as highly polymorphism and reproducible hence it consider extremely useful in creating quick genetic map and transcript profile, AFLP Marker is a dominant inheritance marker because it doesn't distinguish between heterozygous and homozygous alleles and generate huge information leading to require technically skills in laboratory and data analysis so it need more time therefore it consider complex and costly marker, AFLP Marker use in genetic diversity studies and germplasm collection studies and genome mapping and gene tagging, AFLP Marker based on selective PCR amplification of digested DNA fragments after digest DNA and ligate to synthetic adaptors and amplifying by primers that carry selective nucleotides at their 3 ends to detect polymorphism by changes in restriction sites or their neighboring regions, AFLP Marker detect genomic restriction fragments using PCR amplification for detection restriction fragments to display presence (1) or absence (0) restricted fragments while RFLP Marker detect length of restricted fragments using southern blot hybridization to detect restricted fragments to display length differences of restricted fragments (Watch Related Video in #geneticteacher) #geneticteacher