3. You are a process engineer at JlumoiaSequencing and are in charge of making s

Question

3. You are a process engineer at JlumoiaSequencing and are in charge of making sure sequencing runs go smoothly Describe the step-by-step method for how sequencing by synthesis with reversible chain terminators. Indicate what and the template strand, growing strand, polymerase, and bases are using the diagram below (next page) a. b. What direction does the growing strand elongate (the strand to which the reversible chain terminators are added)? Your intern informs you that one of your sequencing machines is spitting out some funny data. They confess that they added the standard reversible chain terminators for A, G, and C bases, but accidentally added a non-reversible dideoxythymidine. What do you expect the sequencing data to look like under these conditions and why? c.

Explanation / Answer

Sequencing by synthesis has four fluorescent labeled nucleotides which are used to sequence millions of clusters on the flow cell surface in parallel. A single labeled dNTP is added to the nucleic acid chain in each sequencing cycle, which serves as a terminator for polymerization. It so happens that after each time dNTP is incorporated, the fluorescent dye is made to image, which helps them to identify the base. It is followed by the enzymes which cleaves the next nucleotide.The 4 reversible terminator-bound dNTPs are present as single, separate molecules. In the end result is obtained highly accurate base-by-base sequencing which does not have sequence-context specific errors.

Reversible chain terminators are added in the direction 5' to 3'.

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