There are two sequencing-by-synthesis methods (pyrosequencing and illumina rever

Question

There are two sequencing-by-synthesis methods (pyrosequencing and illumina reversible terminator methods) as well as the older chain termination method with fluorescently-tagged dideoxynucleotide terminators(sanger sequencing). During the individual sequencing reaction & data collection for these methods, there are fundamental differences in expectations because of the methods' properties, although the data analysis will produce the same final DNA sequence for the template if all goes well.

Compare and contrast in depth (evaluate) the 3 methods (pyro, illumina, and sanger) for the biochemical components/ reaction in template sequence determination (at the nucleotide level) and detection of that result. Do not just give an explanation of the three techniques. Point out what is similar or different between the techniques and explain those aspects.

For example: In the two sequence-by-synthesis methods, the oligonucleotide primer that is used as the starting point for the polymerization reaction in sequencing must be complementary to a sequence that was added as an adaptor to the template DNA because the template is generated from sheared DNAs and is itself generally unknown. In contrast, because the templates in Sanger sequencing are clones or PCR products, the sequencing primers are designed from prior sequence information about the input template DNA.

Explanation / Answer

Sanger sequence method can be used to sequence long DNA sequence at once and is very accurate while pyro and illumina uses short DNA sequences each time to sequence the entire DNA and is very accurate as well. Sanger sequence method requires pure DNA for sequencing that may either be cloned or PCR amplified while pyro and illumina methods use short sequence of DNA mixtures to which adapters are ligated and each DNA fragment is considered separately for sequencing.They do not require cloned or PCR amplified DNA. In sanger method, corresponding to each base, we can get a mean peak value from the chromatogram while in pyro and illumina methods we get individual peaks for each DNA molecule that is sequenced separately. In sanger method, the first results from beginning 20-40 bases are not of good quality but after that and upto 1 kb the results are good while in pyro method from first to 80 bases, the quality of the results are good.

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