14 replies to this topic

[Ameya P]

HI all,

Have been going through a few papers (1988-1993). I would like to repeat a few experiments from there. However, all papers mention that to sequence the genes of interest, they amplified the DNA using specific primers and cloned the PCR product into some phage DNA, before it was sequenced.

Is it necessary to clone the DNA before sequencing, even today??? Would they then do it because that sequencing techniques were not well developed???

Thanks

Edited by gt_ameya, 13 September 2010 - 09:51 PM.

[Rsm]

No, you don't need to clone before sequencing. Run your PCR, check your product, take 1ul directly for the sequencing reaction (no clean-up necessary if you have good product). To clone it beforehand improves the quality and length of your sequence read, but is not necessary per se.

rsm

[HomeBrew]

I disagree with Rsm (sorry)...

Sequencing a PCR reaction directly and sequencing a fragment cloned from a PCR reaction gives you two different pieces of data, which may or may not be the same.

When you sequence a PCR reaction directly, you get back the sequence of the majority of the population of amplicons, whereas when you sequence an individual cloned PCR amplicon, you get back the sequence of that particular amplicon. Due to the possibility of base incorporation errors during amplification, the sequence of the cloned amplicon might differ from that of the population.

As an example, let's say you have 1,000 amplicons of a 1,000 basepair fragment. Let us further say that each one of these amplicons has one base incorporation error, and that they're all in a different location. If you sequence such a reaction directly, you'll get back the true sequence of the fragment, because the single error in each position is overwhelmed by the correct base that exists in the other 999 amplicons. From this population, however, it would be impossible to clone a fragment that has the correct sequence.

It's like saying in a classroom, most students have brown eyes. But if you pick an individual student, he or she might have blue eyes -- you won't know until you look at the student you've picked what color eyes he or she has...

If your goal is to determine the sequence of a fragment as it exists on the chromosome, then sequencing the PCR reaction directly is okay. If your goal is to clone a fragment from this population and use it in further studies, you need to sequence the individual clone to insure it has no base incorporation errors.

[Adrian K]

not forget to mention, if you sequence your pcr directly, usually the first 40-100 bases will not be accurate or correct.

[Ameya P]

Thanks Hone Brew,

My goal is to determine the sequence of the fragment as it exists on the chromosome. SO I can sequence the reaction directly.

What is the maximum length of fragment that can be sequenced with minimum error??? I am trying to look for SNPs in 5 different exons of a gene. So I have to design 5 primers, PCR and sequence therm individually, right??? or is there a faster way???

Thanks,

[Ameya P]

That information could prove very useful...

not forget to mention, if you sequence your pcr directly, usually the first 40-100 bases will not be accurate or correct.

[Rsm]

not forget to mention, if you sequence your pcr directly, usually the first 40-100 bases will not be accurate or correct.

That depends on the sequencing primer you use. If you have them in the middle of your gene, 150bp apart (say, you used them for qPCR before), then you can sequence your PCR product directly and lose maybe 7 to 10 bases, not more.
Actually, I guess that if you clone your gene, make a nice prep, and do the sequencing reaction with your gene-specific primers, then you'll lose 40-100bp as well. Or can you retrieve the very 5' of your sequence, like immediateley downstream of your primer? I wouldn't think that's better if you made a prep.

rsm

[Adrian K]

in other words, if you so not wish to lose a single base, clone it into a vector (blunt end or TA cloning), sequence it by using the vector's sequencing primer (T7, M13 etc...).

How long is your fragment (estimate)?

[Ameya P]

in other words, if you so not wish to lose a single base, clone it into a vector (blunt end or TA cloning), sequence it by using the vector's sequencing primer (T7, M13 etc...).

How long is your fragment (estimate)?

Ideally, I would like the whole gene sequenced (its like 5 exons, 50 kb). Splitting up exons, I get a PCR fragment around 400bp. Now, if the first, say 50 bp, are going to be incorrect, then I have design newer primers say 100bp upstream of the primers I have right now. So, I was wondering if there's a faster way of doing this. Also, getting the whole gene sequenced would give me loads more information, I could work on.

[Rsm]

Ideally, I would like the whole gene sequenced (its like 5 exons, 50 kb). Splitting up exons, I get a PCR fragment around 400bp. Now, if the first, say 50 bp, are going to be incorrect, then I have design newer primers say 100bp upstream of the primers I have right now. So, I was wondering if there's a faster way of doing this. Also, getting the whole gene sequenced would give me loads more information, I could work on.

Most (of my) sequence reads are 700-850bp of good quality, so you'll need a sequencing primer every 500 bases. For 50kb that would be 100 sequences. That's quite a lot... Maybe you better focus on some specific region.

Cheers,

rsm

[HomeBrew]

There is difficulty at both ends of a sequence run: the 5'-end has problems for a short period, and the 3'-base calls get progressively weaker as the sequence extends. The solution is to sequence each clone in both directions, preferably using primers that bind to the vector about 30 bp to either side of site into which your fragments are cloned. With ~400 bp fragments, you'll get complete coverage...

[Ameya P]

Thank you all for the advice.

Since, I am basically looking to screen SNPs, I am thinking of sequencing the PCR product directly. It will be quicker and will also give me what I require.

Now to do that, do I need a different primer for sequencing purposes??? or can my PCR primers do the job?

[Trof]

Sequencing read length depends mainly on the instruments and chemicals used. For example ABI instruments can have capillaries of various length, various types od polymer inside, so you may get up to 700-800 bp reads or only around 300-450. In shorter reads less bp ale lost on the beginning, also ABI v1.1 kit is better than v3.1 if you want to read closer to the primer.

Once you know how long reads you will get and what type of sequencing kit you are gonna use, then you can design your primers. For example if you know you will get good 450 bp sequence, starting 40 bp from the primer, then you design forward primer 45 bp upstream of beginning of the exon and make maximum 900 bp amplicon to sequence using only PCR primers. If you have longer amplicon, then use inner sequencing primers. You can derive your amplicon from DNA having primers in introns, if exons have suitable length and numbers (5 exons is not much, so I would recoment this, one amplicon on each exon). Other way is to transcribe RNA to cDNA and amplify the whole coding region, this is practical if there are lots of small exons and overal length of coding region is up to say 1kb (and if you have the RNA of course). So this also depends on your gene.

[mdfenko]

Now to do that, do I need a different primer for sequencing purposes??? or can my PCR primers do the job?

you can, but it is not recommended. sequencing primers may be longer than pcr primers (~30 bases). they should be cleaner. you don't want to start from the very end of the fragment to avoid slippage.

Edited by mdfenko, 17 September 2010 - 07:56 AM.

[Stephbaker]

I found some interesting and useful stuff here also.