Which of the following is NOT required in a Sanger sequencing reaction?
A standard Sanger reaction requires a single-stranded DNA template, a complementary oligonucleotide primer providing a free 3' hydroxyl, DNA polymerase, all four deoxyribonucleotide triphosphates, small amounts of dideoxynucleotide triphosphates, and magnesium-containing buffer. The polymerase extends the primer synthesizing DNA complementary to the template. RNA template is not required because the technique directly sequences DNA; if RNA were the analyte, it must first be converted to cDNA by reverse transcriptase. Therefore DNA polymerase, ddNTPs and primer are indispensable, whereas RNA te
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.