Question

Now consider a sequencing method where bases (A, T, C, or G) are each “read” as a polymerase incorporates them cycle by cycle into a template strand of DNA. Many strands of DNA are sequenced simultaneously.

- During each cycle, the polymerase will incorporate either one nucleotide or no nucleotide, and fresh polymerase is supplied after each cycle (so you do not have to worry about enzyme lifetime). The probability of incorporation is called the efficiency of the polymerase.

- The probability that an incorporated nucleotide is correct is the polymerase accuracy. The probability that an incorporated nucleotide is incorrect is the polymerase error rate.

Readout: The readout for this method is fluorescence. Each base is labeled with a different color, but to “call” the correct base during each cycle, at least 50% of the DNA strands must contain no mutations (at all). What is the maximum theoretical read length that could be used for this method?

Answer 1

This type of sequencing is a higher volume sanger sequencing has been replaced by the next gen methods, especially for large scale automated genome analyses. However the conventional sanger sequencing method is widely used for small scale projects, validation of Next gen results and obtaining especially long contiguous DNA sequence reads which are >500 nucleotides.

Using fluroescence based sanger sequencing method high quality sequence for relatively long stretches of DNA upto 900 nucleotides can be sequenced.