Mate-Pair biotinylation

[djlarsen]

Hello, I'm trying to make a "homemade" set of reagents to perform the Illumina mate pair library prep. However, I can't quite seem to find any definitive info on the 1mM biotin dNTP Mix used for end labeling. I've seen biotin dNTP mixes that have every dNTP biotinylated, some have one nucleotide labelled while others have fractions of nucleotides biotinylated(say 1/3 of all dATP's are biotinylated) Does anyone have any insight into this?

[thesmurph]

Hello. I was wondering if you had had any advances on your "homemade" mate pair reagents? I too am venturing into the realm of homemade reagents and would appreciate any advice on: Biotinylation/Intramolecular-Cirrcularization Enzyme/Exonuclease Enzyme

Biotinylation: Do you need all nuc's biotinylated in the 1mM mix or is single Bio-dATP adequate to get end biotinylation? What source?

Exonuclease: NEB Lambda Exonuclease?

Steptavidin Beads: Invitrogen Dynabeads and buffer system?

Would appreciate any advice

Always easier to jump on a work in progress than start from scratch : )

[djlarsen]

from the research I've done on end labeling, it sounds like only one dNTP is typically labeled. The other three are standard dNTP's. I'm currently leaning towards using the neb dntp mix with a fraction of the dATP biotinylated--but I haven't put this into action yet.

Exonuclease-probably lambda exo

Strep beads-invitrogen

circularization ligase- probably T4 ligase

[thesmurph]

Thanks for reply. That is what I also suspected with the biotinylation. In the Illumina MP library prep they have now combined end repair/biot'n steps. They now spike in the Biotin dNTP after a pre-15 minute end repair incubation with no clean up in between. I thought this would be more efficient to just spike in single bio-dATP and/or bio-CTP (invitrogen). Why would they still apply a mixed dNTP/bio-dATP mix?

The mechanism appears to be a nucleotide replacement mechanism near the end of the molecules. So would you expect multiple biotinylation?

For the circularizing enzyme how would you prevent inter-molecular ligation with T4 ligase? I suspect it will be very promiscuous! It must require a more stringent ligase or is the dilution to 300ul the answer?

[djlarsen]

I hadn't heard they changed the protocol . When I first looked at their protocol, it occured to me that the two step process seemed unnecessary but I didn't want to change something that worked for them.

In the literature Iv've read, you really want as few biotinylated dNTP's added as possible-an increased concentration of biotinylated dNTP's would cause some steric hinderance when binding to the strep beads. I think that's why only a fraction of the dATP's in NEB's mix is biotinylated. As for the mix rather than the single dNTP, it may be to keep an equal concentration of each dNTP--but thats just a guess.

As for the ligase , I think the dilution would be the key. I know my PI used T4 when constructing plasmids so it should be able to circularize them--but if you come across a better product let me know.

[Apratim]

I am also after "Home-made" mate-pair protocol

I am also wondering about the biotinylation step. Also when you pre-incubate with T4 DNA polymerase, T4 PNK, and Klwnow in presence of dNTPS will you not polish/fill the the ends? Then how the bio-ATP is getting incorporated?

Dynabead had M-270 for biotinylated DNA/RNA and I routinely use it for cDNAs for SuperSAGE library preparations. Other option is Dynabead kilobase binder which is optimized for >2 kb fragments. Not sure if that is what Illumina supplies.

As for circularization, dilution is the key. This is routinely done for Inverse PCR.

[djlarsen]

I am also wondering about the biotinylation step. Also when you pre-incubate with T4 DNA polymerase, T4 PNK, and Klwnow in presence of dNTPS will you not polish/fill the the ends? Then how the bio-ATP is getting incorporated?


The DNA fragments are first blunt ended with a preincubation with standard dNTP's and polymerase cocktail. Following this, the blunt ended DNA is incubated with biotinylated dNTP's and the same polymerase cocktail. The polymerases have a exonuclease domain that removes the ends from the blunt DNA fragments and then fills in with free dNTP's and hopefully incorporates one of the biotinylated dNTP's in solution. As for the beads, Illumina asks you to supply your own M-280 Strep Dynabeads to purify biotinylated DNA.

[Apratim]

Thanks for the explanation. I can understand when you are polishing a 3' overhang you can remove extra base by exonuclease activity and then fill-it up and hopefully incorporate a biotinylated one. But cannot it be done at the same time? Particularly when you are filling in a 5' overhang? Is the pre-incubation step must? Also, if you are using only biotin-dATP your chances of incorporation biotin is less than if you use biotin-dNTP. But I suppose your read depth and randomness will account for that.

[grace033]

I think the pre-incubaton step is must ,it can help to repair DNA nick .and biotin-label ,is a chemical reaction of dynamic equilibrium。

[Paulo.jt]

Hi,
Did you set up a protocol for mate-pairs using homemade reagents?
Which bio-dNTP should work?
Looks like people were talking about this one:

http://www.neb.com/nebecomm/products/productN7552.asp

Has anybody ever tried this?

[Protaeus]

Anyone have an update regarding successes/failures as it relates to the homemade Illumina mate pair protocol?

[jprooney]

Any updates on which dNTP mix to use?