Question

Illumina sequencing is a ‘Next Generation Sequencing’ technology. Explain the flow of how the Illumina sequencing technique workk. (1.5 pts)

There are several commercially available DNA polymerases that are used in sequencing. Two of these DNA polymerases are Taq and Q5. Explain some of the similarities and differences between these two polymerases. (1 pt)

Answer 1

Illumina uses a "sequence by synthesis" approach. This process takes place inside of an acrylamide-coated glass flow cell. The flow cell has oligonucleotides (short nucleotide sequences) coating the bottom of the cell, and they serve to hold the DNA strands in place during sequencing.

Taq DNA polymerase. For this reason, Taq is commonly tested side by side with other polymerases in fidelity measurements. For example, using the blue/white method and correcting for nonphenotypic changes and error propagation during PCR (2) scientists at New England Biolabs measured an error value for Taq at 2.7x10-4±0.8x10-4 or 1 per 3,700 bases. Translating blue/white error rates into practical use, these data suggest that after using 25 PCR cycles to amplify a 400 bp fragment with Taq, several isolates should be screened because about half the clones are predicted to have an error. For larger fragments of approximately 1,000 bp, each clone amplified with Taq is likely to have an undesired mutation. In contrast, the ultra-high-fidelity polymerase Q5® exhibits error rates of 100 X lower than Taq. Based on this value for Q5, one can predict that 199/200 clones amplified will be correct.