10 replies to this topic

[Maddie]

HELP. I am looking at library preparation for next generation sequencing but I am lost with the enzymes and their action. To end repair DNA extracts, Illumina uses T4 DNA polymerase coupled with Klenow DNA Polymerase and T4 PNK  
Is there a book or a website that explains CLEARLY what each enzyme is doing and why we need them?

Also, if I did a linear amplification (one primer only) and wanted to cut all the overhangs after that, which enzyme should I use?

I'd highly appreciate your help.

Maddie.

[epibio]

Klenow fills in 5' overhangs (5'-->3' polymerization activity). It also has 3'-->5' exonuclease activity (to remove 3' overhangs) but it is relatively weak in comparison to T4 DNAP. Therefore, T4 DNAP is used to remove 3' overhangs.

The T4 PNK step phosphorylates the 5' ends of the blunt-ended DNA, to enable subsequent ligation of adaptors.

A better approach to library preparation, though, is to avoid shearing, end-repair, and ligation altogether.

For your second question, do you mean you are doing a linear amplification of genomic DNA, and then using the amplification products for library prep?

Edited by epibio, 17 June 2010 - 06:44 AM.

[Maddie]

Thank you SO much epibio.

I do not shear my DNA because I am working with very degraded DNA from bones, but why do you say I should avoid the end repair and ligation? How else could I add my adaptors?

You are correct, I would like to use the PCR products + the rest of the extract actually for the library. My problem is that in my extracts, I have very small amounts of degraded human DNA and tons of microbial DNA. I was hoping that several cycles of linear amplifications targeting the human DNA would help increase and create DNA having a better quality, which would ligate the adaptors better. Problem is I may end up with very long fragments of ssDNA on the 5' end. Could Klenow deal with that or is it usually used to fill up small gaps?

[epibio]

Sounds like you're doing a variation of primer extension capture. I wonder if an array-based target enrichment method may work better. This approach was used, for example, in the recently published sequencing of the Neandertal genome.

Anyway, my original comment about avoiding shearing/end-repair/adaptor ligation is that there are multiple steps involved, while the Nextera method combines those into a single reaction. It's probably not suitable for degraded DNA, though.

Klenow is typically used to fill in small overhangs, but it should still work in your case.

[Maddie]

epibio, on Jun 17 2010, 12:35 PM, said:

Sounds like you're doing a variation of primer extension capture.

That's exactly what I am doing ..or planning on doing. I noticed that degraded DNA does't do good with library prep so I was hoping to enrich first (and then again later after amplifying the library)

epibio, on Jun 17 2010, 12:35 PM, said:

I wonder if an array-based target enrichment method may work better. This approach was used, for example, in the recently published sequencing of the Neandertal genome.

No money for that  

epibio, on Jun 17 2010, 12:35 PM, said:

Anyway, my original comment about avoiding shearing/end-repair/adaptor ligation is that there are multiple steps involved, while the Nextera method combines those into a single reaction. It's probably not suitable for degraded DNA, though.

I looked at the site. Their kit looks very cool and I did write to ask if I could avoid the shearing part. No can't do.
I'll have to do each step at a time.

epibio, on Jun 17 2010, 12:35 PM, said:

Klenow is typically used to fill in small overhangs, but it should still work in your case.

How long should I incubate, you think?

[epibio]

Quote

How long should I incubate, you think?

One hour at 37C should be more than sufficient, but I'd suggest using the exo minus mutant rather than "standard" Klenow.

[Maddie]

So, you would mix the mutant klenow with the T4 DNA polymerase? I may have overhangs on the other side too (especially if I have damages or abasic sites that stop the elongations).

[epibio]

Yes, that should be fine. You can Google "end-repair" for reaction conditions, or purchase kits where the conditions have been already optimized.

[Maddie]

Cool. What about the abasic sites in the middle of the fragments? What enzyme do I need there to fill up gaps?

is that the baby I need?
http://www.neb.com/n...roductM0212.asp

Edited by Maddie, 17 June 2010 - 01:15 PM.

[phage434]

There are enzyme mixes intended to fix up degraded dna, such as the epicentre pre-pcr mix, and equivalent kits from neb.

[Maddie]

Thanks. I should receive a kit soon.  

Does anyone has experience with the preCR kit of NEB?