DNA sequencing. Fredrick Sanger invented the first method for sequencing DNA, wh

Question

DNA sequencing. Fredrick Sanger invented the first method for sequencing DNA, which took advantage of knowing the mechanism of DNA polymerase. His method utilized 2', 3'-dideoxynucleotide triphosphates as chain-terminating substrate analogs. What substrate analogs could be used to sequence RNA using RNA polymerase? A DNA was sequenced using the original Sanger method. The sequencing reactions were analyzed by gel electrophoresis (shown below). Each reaction contained a different substrate analog (labeled in the gel lanes as A, G, C, and T). A primer was paired with the template strand of the DNA. Marked on left side of this gel is the gel band of radioactively labeled primer. In the space provided below, complete the base sequence of the template DNA strand.

Explanation / Answer

A) There are very few photoactivatable nucleotide analogs that are available as phosphoramidites or function as substrates for bacteriophage RNA polymerases, and most of them are pyrimidine-based.

B) Template strand: 3’-CGAAGGAG; its complementary base strand is: 3’-GCTTCCTC

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