[Ghaz]

Is it possible to use PCR in DNA sequencing ?please justify.

[sp_08]

it might be possible to obtain the sequence through pcr but you will not be able obtains the correct order of the genes, like which gene occurs before and which one occurs later...

[CharonY]

Read up on Sanger sequencing.

[a_j]

PCR is a necessary part of Sanger sequencing but I would say that it is not possible to sequence with PCR. What u know is that the primer attaches at a certain sequence but have to know this sequence first to design the primer..

[CharonY]

So how does the Sanger sequencing initiate?

[Ghaz]

People its not getting clear to me

[Arete]

Ghaz, on 24 January 2012 - 02:28 PM, said:

People its not getting clear to me

http://lmgtfy.com/?q=sanger+sequencing

For those reading along - Invitrogen Ion Torrent technology is looking promising for benchtop next gen sequencing, but isn't PCR. I'd be considering it if I wasn't using crappy fta card samples and trying to pull parasite DNA out of vertebrate blood samples.

http://find.lifetech...om/ionrnaseq/fl

[CharonY]

Well, most (all?) next-gen sequencing systems rely on PCR. The Ion torrent uses ion semiconductor sequencing technology, which is very similar to pyrosequencing. The main difference is that detection and subsequent base calling is not based on optical data (i.e. fluorescence) but by detection of H+ ions. PCR is still there for building in nucleotides.

[Ghaz]

Dont confuse me with high fi things....Be precise and give to the point answer.

[Arete]

Ghaz, on 24 January 2012 - 05:24 PM, said:

Dont confuse me with high fi things....Be precise and give to the point answer.

The question as posed is nonsensical. Read up on Sanger sequencing and once you understand the process, come back with a sensible question we can provide a sensible answer to - otherwise expect disappointment.

CharonY - I freely admit to being an end product user and not as clued in to the tech as I should often be- thanks for the clarification. We just sent out some samples for PacBio RS SMRT™ (Single Molecule Real Time) *take a breath* sequencing. The strobe sequencing method offered by our core offers some pretty amazing read lengths from our typically crappy template - 6kb average reads and 35mb coverage from one lane. http://www.pacificbi...es.com/products

[CharonY]

6 kb is really nice. Read length is actually one of the biggest issue with most next-gens are relatively short read lengths. Many are aimed at massive parallelization. But with the arrival of high-sensitivity monitoring together with submicrolitre volume handling the traditional approaches can be happily married with traditional enzymatic assays and fluorophore detection, such as the PacBio system. Some are exploring alternative approaches that, in theory, allow the sequencing of a complete DNA molecule in one go in enzyme-free systems, but I still more skeptical regarding those.

I am not doing any real sequencing any more (moved on to way more downstream stuff), but I can say that from a technical and especially price viewpoint the technologies available are quite exciting.
Though as a biologist, I am more stumped with what to do with all the data .

[Arete]

The PacBio super long runs seem to be highly prone to incorrect base calling (like 15% error rate). So for SNP calling, they're no good. What we're using them for is to try and get at something short read referenced alignments can't get at - complete loss of functional genes and variation due to indels.

It's no silver bullet but it's another tool in my exponentially growing and bewildering toolkit

[CharonY]

Oi, 15% is rather bad for de novo sequencing (something I am more interested at). Guess I am stuck with 454 or solexa for the moment. Having a wide range of tools is nice. Although I learned that for getting a job it is sometimes better to reduce the range as not to confuse the search committee.