Sequencing:why do pcr AND cloning into plasmid? - (Mar/05/2008 )

I want to sequence a certain area, I was planning (and am actually doing) this by using PCR to amplify the 2kbp fragment and sequencing inside it. I just saw a paper that does some bisulfite treatment (to see which nucleotides are methylated or something) and then uses PCR to multiply out a fragment (of roughly 200-400bp), then clones it into a plasmid, then sequences it.

I don't understand? Is it because the fragment is small? Is it easier than doing a larger PCR fragment (I know PCR of a smaller product is easier in most cases)? But I thought cloning into a plasmid was difficult?

???

---

by putting the pcr product into a plasmid you can use a standard primer and sequence the entire product. if you just use the pcr product then you will not get the sequence for the primer side end (which will probably be noisy).

---

I don't fully understand what you said. So if I PCR from say nt 100-2100 and then sequence using a primer inside that region (say at 200) then I only get the sequence from 200 on, right? Why not then do the original PCR from 1-2100 and use a nested primer at 100 for sequencing if you want that area?
What did you mean by 'which will probably be noisier'?

Thanks for the help.

---

you will not get the sequence from 200 (from your example). the primer starts at base 200 and ends at base 218-230 (depending on the length of the primer). then you aren't actually able to read the sequence for another 5-20 bases (depending on cleanup method and efficiency). so you may not start seeing reliable sequence until base 250.

if you insert the pcr product into a vector and start sequencing from within the vector then you can get sequence from base 1 of the pcr product (find the insertion point and take sequence from there).

the noise to which i referred is from the inability to discern clean sequence near the primer (interference caused by dye peaks and saturated fragment response (too many small products from the sequencing reaction)).

---

Thankyou for clarification. That's what I thought you meant. But then, most of the time I walk around thinking I understand something, only to find out later I'm wrong .

Thanks

I still wonder - why not then pick the intial primers further in front (like for the 200 example, if the primer's at 100-130 then you get from 140 on - so you have your sequence clear starting at 200)? Isn't plasmid work harder than an easy ordering of oligos and pcr reaction?

---

Another reasion might be the bisulfite treatment efficiency, if the efficiency is not 100%, therefore, seqeuncing PCR products would show merged peak, however sequence of cloned fragment will show clear peak for identified base...

---

in the paper you saw they probably wanted the complete sequence of the pcr product so they would have to start sequencing some number of bases prior to the insert.

since you want to sequence inside the pcr product then you do not need to insert it into a plasmid vector unless you need to sequence too close to the primer.

and, yes, sequencing of a pcr product is usually easier than sequencing a plasmid (especially with todays capillary electrophoresis based sequence analyzers, or, at least, the ones from beckman-coulter).

---