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

DNA sequencing is a method used to detemine the sequence of nucleotides Adenine, Guanine, Cytosine, Thymine in a particular region of DNA.

Sanger sequencing

Chain termination method was developed by Fredrick Sanger and so the method is called Sanger sequencing. This method was used in the first generation of DNA sequencers. Sanger sequencing assay accurately reads a maximum of about 700-800 bp in length.This method is based on the use of chain terminators, the dideoxynucleotides.The dideoxynucleotide differ from deoxynucleotide by the lack of a 3'OH group on the five carbon sugar.

Steps in Sanger sequencing:

Pyro sequencing

This method is also used for the determination of order of nucleotides in DNA based on the principle of "Sequencing by synthesis".In Sanger sequencing, DNA sequencing is based on the selective incorporation of chain terminating dideoxynucleotides but in Pyrosequencing it depends on the detection of pyrophosphate release on nucleotide incorporation.

Illumina sequencing

This methd for the determination of DNA sequencing is based on the reversible dye-terminators that enables the identification single bases when they are introduced into DNA strand. Illumina uses a "sequence by synthesis' model.

DNA sequencing technology have gone through three generations, Sanger sequencing is first generation, Pyrosequencing is second generation and Illumina sequencing is next-generation.

In sanger, only low number of samples can be analysed.

In pyro, DNA sequencing of up to one hundred base pairs is done. It avoids the need of labelled primers, labeled nucleotides.

In Sanger sequencing, a reading gap of roughly 20-30 bases from the sequencing primer but in pyrosequencing can generate sequence signals immediately downstream of the primer. As sequencing starts with the first base next to the annealed primer, making primer design becomes more flexible in this method.

Pyrosequencing differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides.

Since there is no chain termination in pyrosequencing other than by designed unavailability of the other 3 nucleotides, pyrosequencing reads have insertion/deletion errors particularly in or next to runs of homopolymers.

In principle Sanger sequencing requires a pure DNA sample (whether cloned or PCR amplified) with a possible primer binding site. the primer should be amplifying a specific product which gives the sequence, so very often cloned DNA / gel purified PCR products are necessary.

The Pyro and illumina sequencing approaches usually use short DNA sequence mixtures, ligate adapters and consider each DNA fragment separately for sequencing. This makes sequencing of a mixture of DNA possible, avoiding cloning, only limitation being the read length.

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