How to eliminate PE dimers from final product?

[OnUrMark]

Greetings RNA-Seq'ers,

I am preparing samples for RNA-seq, and my final PCR enrichment product contains dimers and trimers of my 350bp fragments (~700bp and ~1000bp). I have gel extracted the PE product at 350bp, but the BioAnalyzer still shows the the dimers and trimers. Does anyone know how it is possible that the longer fragments are running at the same speed as the 350bp fragments on the gel? Any ideas on how I can remove the dimers from my PE product?

Thanks a Bunch!

Melanie

[genlyai]

Hi Melanie,

I've never seen this with this process, but in principle, it's possible to get hybridization (rather than covalent) concatamers in this system. This can happen in a highly multiplex PCR where all the PCR products have the same ends but different inserts.

If you denature your products at any point (for example if you overcycle the PCR), they are unlikely to find their correct partners back, but the adapters are indeed likely to hybridize. This will result in something that is double-stranded on one end, but single stranded through the insert region. From here, it can go one of two ways: either the adapters at the other end can hybridize to each other, making a complex that should run at roughly 350bp; or they can hybridize to adapters in other molecules, making a complex that can run higher. Compared to normal PCR, this second possibility is favored in the multiplex case because the original hybrid cannot "zip up" through the insert region (processivity of hybridization is lost). Obviously, the second case also becomes more likely the more concentrated your material is.

Anyway, not sure how likely this is in your situation. My main concern is that the higher MW hybrids would not necessarily be expected to run at even multiples of the monomer. If I am right, however, you should see 2 things:

1. The material runs as a unique band if run on a denaturing gel.
2. The problem goes away if you take care not to denature your product. This probably just means fewer PCR cycles.

Let us know how this turns out for you.

[OnUrMark]

Thanks for your explanation and ideas. Today I had several samples denatured before they were run on the bioanalyzer. The 700 bp peaks did disappear for all samples, and all samples had peaks only at 350bp.

Can you help me with the nitty gritty details? I am having difficulty understanding what the longer fragments look like. What is linked to what, why will one end be double-stranded, and why will they return to 350bp when denatured before running on a gel?

I believe I am using very few cycles (10-15), and I still see the 700bp peaks on 10cycle samples. After P.E. I gel extract my 350bp product before checking it on the bioanalyzer and sequencing it on the illumina. The first step is to dissolve the gel in buffer at 50C to melt the gel slice. Could this be the step that denatures my product and then they hybridize after cooling?

Also, would more primer during P.E. be another solution to possible hybridization during pcr? (I'm trying it right now, and will post the results.)

Thanks Again,

Melanie

[HESmith]

Denaturation during 50C gel solubilization is likely the problem. Most recent protocols use room temp instead.

Genlyai's description of the products is correct. The ends of your samples are complementary and will base-pair; the inserts are unique and will not. Multiple ends can anneal to form daisy chains. Try drawing a diagram to visualize what's happening.

The good news: as long as the library has the appropriate adapter ends, it's fine for sequencing (it gets denatured to ssDNA before loading). The only challenge will be to accurately quantify your library concentration.

[OnUrMark]

Thank you for your help on this problem. I will perform gel extraction at RT from now on, and it makes sense that this will alleviate the problem.

I understand Genlyai's description of the complementary adapters with different inserts pairing up, but I don't understand how one fragment would be double-stranded while the other would be single stranded (unless that only happens with over cycling pcr). It seems that the daisy chain formed would be single stranded. I often see a peak at 950bp representing three fragments linked together. My diagrams of di and tri hybrids are all single stranded.

Thanks So Much!

Melanie

[genlyai]

I agree with the above. Let us know if you need further clarification.

HESmith is also right that it should be fine for sequencing, but problematic for quantification by picogreen or A260. A PCR-based quant like Kapa's kits should still be fine.

[HESmith]

Thank you for your help on this problem. I will perform gel extraction at RT from now on, and it makes sense that this will alleviate the problem.

I understand Genlyai's description of the complementary adapters with different inserts pairing up, but I don't understand how one fragment would be double-stranded while the other would be single stranded (unless that only happens with over cycling pcr). It seems that the daisy chain formed would be single stranded. I often see a peak at 950bp representing three fragments linked together. My diagrams of di and tri hybrids are all single stranded.

Thanks So Much!

Melanie

350bp = one ssDNA molecule; 700bp = two molecules, ds at adapter end, otherwise ss; 1050bp = three molecules

[OnUrMark]

Ok, thanks. I was misunderstanding what part of the hybrid was ds.

[shoegame2001]

I am having this same issue of concatamers forming after I perform gel extraction, since the protocol involves melting the agarose at 50C. Does anyone know if there is a temperature/time that I could use to still solubilize the agarose but not denature the ds cDNA? Thanks!

[HESmith]

The answer to your question is among the entries in this thread.

[shoegame2001]

Sorry, I might still be missing something, but the sequencing lab that I am using wants me to rerun the gel and clean it up without producing the concatamers (even though it would probably be fine for sequencing). I was wondering if I could avoid buying a low melting temp agarose, but maybe that is the only option. Or will a normal agarose gel actually dissolve at room temperature in a Qiaquick buffer, for example? Thanks for your help.

[pmiguel]

If you add additional chaotrope, agarose will dissolve at room temp. If you are using 2 volumes of whatever the "buffer" is called to dissolve your gel slice, try 3 volumes.

You may be able to avoid re-purifying your library altogether, though. If you had previously done a PCR amplification on this library, then you could just denature it and allow it to anneal under more stringent conditions. I think we use 90 oC for 2 minutes followed by turning the thermal cycler off, rather than having it quickly ramp back to room temp. This way there is plenty of time for full-length strand annealing to occur before the temps are low enough for the strand-end-only interactions that produce this "daisy chaining".

Note added Feb 12, 2012: So, I think this should not work well in most cases. The problem is that the complexity of the inserts is much, much higher than the complexity of the ends (adapters) with most libraries. To get it to work, you would have to hold the temps above where the adapter-adapter annealing alone is stable, and give the library molecules time to find their complement strands. I don't have the fortitude to dig up the hybridization kinetics formula to do the calculations, but my guess is that we are talking days, not minutes, for this to happen.

--
Phillip