Principle of CAPS and dCAPS Markers

Sanger Sequencing Method or Chain Termination Sequencing Method is first generation sequencing method use Dideoxy nucleotides (ddNTPs) that lack (3-OH) hydroxyl group and incorporate with growing DNA strand during PCR reaction as DNA chain terminators to generate fragments with ends ddATP / ddGTP / ddTTP / ddCTP, dNTPs contains (3-OH) hydroxyl group while ddNTPs contains hydrogen instead of (3-OH) hydroxyl group, Manual Sanger Sequencing Method initiate by DNA extraction and denature DNA to obtain single DNA fragments and divide DNA sample into four tubes with primer and dNTP, and Taq DNA polymerase and one ddNTPs in each tubes to terminate DNA synthesis at specific base to create set of DNA fragments with different length after that separate amplified fragments in parallel lanes (A / G / C / T) in denature polyacrylamide gel electrophoresis and autoradiography gel in X-ray film or UV light to visualize length of amplified fragments and read bands from bottom to top according to direction of DNA synthesis (5’→3’) to compare sequence with reference sequence, Automatic Sanger Sequencing Method fluorescently label primer at 5’ end to amplify specific DNA regions and label each of ddNTPs with fluorescent dyes and run reaction in single lane in denature polyacrylamide gel electrophoresis or Capillary gel to visualize nucleotides peaks that reflect sequence of chain termination to compare output sequence with reference sequence, Sanger Sequence Method consider gold standard for single genes or small DNA regions with base accuracy 99.99% up to 1000bp in single run without require complex bioinformatics tools and experts, Sanger Sequencing Method consider labor and costly and time consuming method and require relatively high quality of DNA samples leading to difficult to apply large scale sequences, Sanger Sequencing Method use in Mutation Studies and Epigenomic Studies and Medical Studies and Pharmagenomics Studies and Forensic Studies, Sanger Sequencing Method use single strand DNA for sequencing and primer to flank regions of target sequence and Taq DNA polymerase to synthesize new DNA strand by adding nucleotides to primer and dNTPs that add in synthesis process of DNA strand and ddNTPs to interrupt DNA strand synthesis as chain termination strand (Watch Related Video in #geneticteacher) #geneticteacher