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

PYROSEQUENCING:

1) In this method single stranded DNA template, primer, DNA polymerase, ATP sulfurylase, luciferase and apyrase are the materials required.

2) Incorporation dNTPs release phyrophosphate. ATP sulfurylase converts PPi to ATP in the presence of adensonine5-phosphosulfate.

3. This ATP acts as substrate for luciferase to convert luciferin to oxyluceferin that generates visible light which is detected be camara.

4. Unincorporated nucleotides and ATP are degraded by apyrase.

ILLUMINA DYE SEQUENCING:

1) This method is besed on reversible dye-terminators which enables identification of single base introduced into DNA strand.

2. Tagmented DNA is produced by enzyme called topoisomerase and adapter.

3. In the next step is reduced cycle amplication. In this process primer binding, indices, terminal sequences are added. This is common in Pyrosequencing method. Indices allow to run different samples together.

4. Illimina uses a sequence by synthesis.approach. which takes place inside of acrylamide-coated glass flow cell.

5. Newly produced double stranded DNA is denaturedm one oligonucleotide will be reverse and other forward for amplication This process is called bridge amplification.

6. Primers are attached to forward nucleotide and four coloured fluorescently tagged nucleotides to the DNA to produce different coloured DNA fragments.

SANGER SEQUENCING:

1. This is chain terminating method requires single stranded DNA, primer, DNA polymerase, normal dNTPs, modified ddNTPs

2. In this method ddNTPs are fluorescently labelled for detecting automated sequencing machine.

3. It is low cost and efficient to obtain longer DNAs. Microfluids allows faster, cheaper easier sequence assembly.

Illumina sequencing method had advange over sangers. Automotaed illumina dye sequencing is possible to sequence multiple strands at once quickly. Disadvantage of this method is enzymes used for this method are expensive.

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