DNA sequencing by the Sanger method takes advantage of which propey of DNA synthesis to generate a sequencing ladder?
Addition of nucleotides requires a free OH group on the 3' end of the DNA strand The Sanger method of DNA sequencing is also known as chain termination sequencing. In this method, DNA strands are synthesized in the presence of a low concentration of dideoxynucleosides (ddNTPs) in addition to deoxynucleosides (dNTPs). Incorporation of a dNTP allows synthesis to continue as there is a free 3'-OH group for addition of the next nucleotide. Incorporation of a ddNTP terminates synthesis of the DNA strand as there is no free 3-OH group for addition of the next nucleotide. In Sanger sequencing, four reactions (A, C, T, and G) are run, each containing the appropriate ddNTP. The mixture of DNA strands generated in each reaction includes strands that are terminated at each given nucleotide (e.g., the A reaction contains DNA strands terminated at each A). The four reactions are run adjacent to one another on an acrylamide gel to separate the strands by size. The sequence of the DNA is determined by reading the order of the terminated chains going up the gel.
Addition of nucleotides requires a free OH group on the 3' end of the DNA strand
DNA has a free 5' phosphoryl group
DNA polymerase has proofreading capabilities
Nucleotides are linked by phosphodiester bonds