Hi Basak,

Never heard of that! What is it good for?

Tom

Oh, it is good for many purposes, usually people use it to amplifiy from known to unknown regions in chromosomes, phage, plasmids, large PCR products and all possible DNA sources, using low stringency. Or, when you want to sequence a piece of DNA in just one direction, for example, you use only one primer.

What I tried to do was a bit different. I linked an RNA oligo to the 3' OH ends of total bacterial mRNA, and used a primer specific to the oligo to initiate RT. Afterwards I used the same primer for PCR (in complementary sequence, for sure). In this was I was hoping to obtain amplified cDNA for my libraries, and since there is only one primer i suppose the amplification would be more linear than exponential... So what I expected to see was a smear starting somewhere from 2000bp to 100bp.

But what I see is unfortunately a very faint smear and two fat bands, one at 200bp and the other smaller. I don't know what the bands are, but I cloned them into a TOPO TA vector, and the cloning worked.. I haven't sequenced them yet, but..

All right, if the bands are single stranded, how should they get into the vector? That is somehow crazy.. and, if they are double stranded, it can only be unspecific binding of something, but I don't know how.

What I am thinking is that, the Taq polymerase is moving at a speed of 1kb per min, right? If you use two primers, the amplification is exponential, so you mainly amplify your desired product. But with one primer, the amplification is linear, therefore the smaller molecules will be amplified faster than the large ones. I thought this is why I might see bands, but I'm not sure, and I don't know how to overcome this problem...

Therefore I thought I could use another protocol that is better to use for a single primer rather than two of them... (Maybe more cycles, less template or less primer?)

I am sorry, this is a bit confusing:( I hate PCR optimization!!

:-)

Your thoughts are quite what I would think. I guess there's no fault in the concept :-)...

Is there literature out there dealing with this stuff?

Basically you've tried whole transcriptome amplification, right?

Just some ideas which you've also had, I bet:

1 Have you checked the linkage efficiency to be sure this worked?

2 What if you ligate an RNA_oligo-PCR_primer-sequence, then go for RT and then for PCR use the PCR_primer for amplification. Maybe the RNA oligo is not perfectly suited as primer sequence for subsequent PCR...

3 What about the faint smere: Wouldn't you expect a faint smere from linear PCR?

(30 cycles: exponential: 1 copy to 1*10e+9 copies vs. linear: 1 to 31 copies)

]:-P fat bands could be some escape strategy for unemployed polymerases :-))

Or could it be - accidentially - that your oligo fitted inside one of the RNA sequences and you had exponential PCR with just 1 primer being forward and reverse at the same time?

4 Couldn't it be that ssDNA ligates into cleaved vector ...? As long as the sticky ends fit?

5 I'm a llllllliiiitttlle bit curious about the sequencing ... =)

6 Regarding 1kb per min: You could use the kits designed for up to 20kb PCR product (Long template ... theres one from Roche.

Keep me updated and keep a stiff upper lip,

Tom

:)) Yeah, the worst is when you get weird results although in your eyes everything makes sense:)

To your first question, I have found one protocol from Cold Spring Harb. Protoc. It is actually really nice, a bit different from what I did -only on DNA and not RNA- :

http://cshprotocols.cshlp.org/cgi/content/full/2008/2/pdb.prot4922

and with one primer:) So i'll definitely give it a try... Yesterday I did another amplification, increasing Tm from 57 to 61, and now I only see a very faint band at 300b, but I see no smear at all...

The bad thing is, there is almost no way to check the ligation efficiency, unless you want to design a whole separate experiment for it (with radio -labeled oligos). What one can to is to get some Dynabeads with complementary sequences to the RNA oligo, and take only the mRNA's that are bound to the oligo from the solution. But if you already have very little starting material, I think it is best to continue in one tube and not introduce too many steps.

I have designed the primers directly on the RNA oligo that I used, so I am sure they should somehow match:)) One mistake I have done was to set the GC content too high (64%), but these double-modified RNA oligos are soooooo expensive (I mean like 400 Euros!!!) therefore I have to make it work, which is a good motivation:))))))

About ssDNA ligating into the vector, I think the ligation efficiency should be very very low with ssDNA, but why not, it should be possible, but I have no experience so far... Ah, another question I wanted to ask, are these fast polymerases reliable if the samples will be used for sequencing later on? I thought they might introduce errors -well, possibly Taq is not thaaat reliable either-

thank you sooo much for the wonderful brainstorming! I'll let you know what comes out of this whole business:))

*smiles*

b.

Yeah, that's right, I just realized it before Easter and hit my head against the walls, for sure it is not possible to prime the PCR from the same priming site as I used for the RT... I will need another oligo annealing step before PCR, I guess, I don't really see how else it is possible to amplify with one single primer and priming site.

tesekkurler:)

basak

Hey,

I wanted to thank you all who helped me out, I managed to correct my protocol and now the whole transcriptome amplification is working perfectly:)

thanks!