Hi,

Immobilization of nucleotides to the Dynabeads can be performed in 3 different ways.

1) Dynabeads M-270 Carboxylic acid (143.05):

amino-oligos can be coupled to these Dynabeads via 5' or 3' amino modification by use of EDC to form an amide bond. The protocol is given in the package insert of Dynabeads M-270 Carboxylic acid.

Using these Dynabeads, one will not have full control over the orientation of the amino-oligo as the coupling may also take place via amino groups of the bases (even though these amino groups are less reactive than the 5'or 3' amino groups) in addition to the one at 5' or 3' site. More control over orientation of oligonucleotides can be achieved by using either biotinylated oligonucleotide coupled onto the Streptavidin beads or covalently binding them to the Dynabeads M-270 Amine.

2) Dynabeads M-270 Amine (143.07)

can be activated with a hetero-bifunctional NHS-ester cross-linker with sulfhydryl reactivity. NHS-ester will react with amino groups on the surface of the beads and leave the sulfhydryl reactive group (Maleimide) free. Using a 5' sulfhydryl modified oligo; the maleimide group will specifically react with 5' sulfhydryl groups and give a correct orientation of primer. Different hetero-bifunctional cross-linkers with long carbon arm can be used for this application all available from Pierce:

GMBS (22309), Sulfo-GMBS (22324), EMCS (22308) or Sulfo EMCS (22307). GMBS has 3 carbon arms and EMCS has 5 carbon arms. Sulfo EMCS and Sulfo GMBS are the water soluble forms.

3) Biotinylated oligonucleotide is the third alternative.

One has full control over where on the oligo the biotin is inserted and hence you will get the same orientation for all the immobilized oligos. The interaction between streptavidin and biotin is the strongest non-covalent binding known with association constant of 1015 M-1.

I hope this will be of help

kind regards

ketil

Hi,

Immobilization of nucleotides to the Dynabeads can be performed in 3 different ways.

1) Dynabeads M-270 Carboxylic acid (143.05):

amino-oligos can be coupled to these Dynabeads via 5' or 3' amino modification by use of EDC to form an amide bond. The protocol is given in the package insert of Dynabeads M-270 Carboxylic acid.

Using these Dynabeads, one will not have full control over the orientation of the amino-oligo as the coupling may also take place via amino groups of the bases (even though these amino groups are less reactive than the 5'or 3' amino groups) in addition to the one at 5' or 3' site. More control over orientation of oligonucleotides can be achieved by using either biotinylated oligonucleotide coupled onto the Streptavidin beads or covalently binding them to the Dynabeads M-270 Amine.

2) Dynabeads M-270 Amine (143.07)

can be activated with a hetero-bifunctional NHS-ester cross-linker with sulfhydryl reactivity. NHS-ester will react with amino groups on the surface of the beads and leave the sulfhydryl reactive group (Maleimide) free. Using a 5' sulfhydryl modified oligo; the maleimide group will specifically react with 5' sulfhydryl groups and give a correct orientation of primer. Different hetero-bifunctional cross-linkers with long carbon arm can be used for this application all available from Pierce:

GMBS (22309), Sulfo-GMBS (22324), EMCS (22308) or Sulfo EMCS (22307). GMBS has 3 carbon arms and EMCS has 5 carbon arms. Sulfo EMCS and Sulfo GMBS are the water soluble forms.

3) Biotinylated oligonucleotide is the third alternative.

One has full control over where on the oligo the biotin is inserted and hence you will get the same orientation for all the immobilized oligos. The interaction between streptavidin and biotin is the strongest non-covalent binding known with association constant of 1015 M-1.

I hope this will be of help

kind regards

ketil

Hi Natascha,

    We routinely capture Illumina libraries with biotin-streptavidin beads that involves wash steps at 65 degrees (biotinylated probes). If this bond was breakable above 45C than all commercial exome and hyb capture methods would be fail (maybe they do to some degree!). I might also be misunderstanding your question. Listen to Ketil he is an expert on all things pertaining to Dynabeads! He has helped me before. Also if you get direct conjugation to work, please share what method you decide on. We are still struggling with the limitations of larger DNA fragment size with streptavidin-DB/biotin pulldown.

Thank you Ketil and Kenneth for your responses. I have been trying to find information about the heat stability of the biotin/streptavidin bond and did not have much success. However I found a paper from 2005 (Holmberg, Blomstergren, Nord et al. Electrophoresis 26: 501-510) that reports about the release % at various temperatures. Figure 3 shows that 50-100% lose the bond when kept at 60°. That got me a little questioning the library preparation... I am just now learning how to do the library preparation and want to understand how it all works.

Natascha this is interesting. Could it have anything to do with the number of biotin molecules proportional to streptavidin beads? We incorporate dNTP-biotin through synthesis etc. At some point we were comparing TdT end labeling to incorporation in the success of pulldowns. One other thing I used to do was to use dual biotin which may strengthen the interaction. 

I think there is a space limit on these response fields...I routinely use MyOne C1 streptavidin beads as it has the highest affinity (Kd=10-15). Maybe fewer probes with fewer biotin molecules may weaken the pulldown. We are now doing washes up to 70C. I could try to find out if we have data on relative yield from expected etc by QPCR. If you describe your experiment I am glad to help troubleshoot if I can...

Natascha, coupling amino-oligos to carboxy-Dynabeads by EDS is quite reliable and old method (Ketil noted it). Sometimes I tried alternative one - I treated amine beads (eg M-270 beads) with some secondary aldehyde (eg glutaraldehyde 0,2% or 0,5% or 1% - optimization needs!) then some (3-4) washes with PBS and mixed with the solution of aminated probes or primers (1uM in PBS); incubation 0,5-1 h and wash. It formed termostable covalent bound primer to bead surfase with aldehyde (carbon) bridge.

Good luck!

Any idea what the maximum density of oligos can be achieved on the Dynabeads?

Ketil Winther Pedersen Dmitry Andreychuk is there any way to immobilise carboxyl functionalised oligo onto amine-functionalised beads? I have been looking around but all the papers and reports mention the other way around, like you have. my oligo probe has a 5'-COOH functionalisation and I want to attach it to magnetic beads (I have both NH2- as well as COOH-beads).

Yes, I have conjugated Carboxyl labeled oligos to amine bearing items. First you need to activate the CO2 using EDC and NHS or sulfo-NHS, then add the amine labeled item. It should work very well.

David E Volk thank you, is there a standard protocol which I can follow in this case?

Check out this site for general info.

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/carbodiimide-crosslinker-chemistry.html

Personally, I'm more of a fan of the Tosylate-functionalized beads. EDC coupling uses harsh reagents that can have unintended effects.

Vincent Paul Schmitz I haven't read about tosylate-based coupling yet, I will read about it. EDC-NHS has been done in our lab for a long time, so I thought I'd also go for it.

David E Volk thank you.

David E Volk are these oligonucleotide functionalized magnetic bead conjugates stable at room temperature?

As stable as any DNA sitting around at room temperature I suppose. Dehydrate them if you are planning to store them a long time without refrigeration.

David E Volk thank you, I was confused about the storage temperature because the beads are stored at ~4 degrees and should not be frozen but the oligos are stored at -20 degrees. So where should the conjugated products go?

Keep them at 4C.

thank you Phillip Kyriakakis

Actually, I have since changed my mind, SuFEx (Sulfonyl Fluoride) click-chemistry possesses the most desirable attributes for conjugating/functionalizing superparamagnetic beads and oligonucleotides. The process is easy, efficient, irreversible, and requires only that the oligos have a 3' or 5' amine.

How to covalently bind Carboxyl-functionalized DNA with an amine functional polymer bead?