DNA sequencing by the Sanger method take: advantage of which property of DNA synthesis to generate a sequencing ladder?: (A) Addition of nucleotides requires a free 01-I group on the 3' end of the DNA strand, (B) DNA has a free 5' phosphoryl group, (C) DNA polymerase has proofreading capabilities, (D) Nucleotides are linked by phosphodiester bonds
Answer is A. 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 deoxynucleotides (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 3OH 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.