PCR on streptavidin coated magnetic beads

[Bluenoise]

Okay, so anyone have any experience with this?

 

Lets say I have steptavidin coated magnetic beads coupled to 500pb Double stranded DNA via biotin. If I wanted to use this DNA as a template for PCR, I am presented with two ways of going about it.

 

1) Eluting the non-biotinylated strand of DNA, and using it as a template for PCR.

2) Using the beads with coupled DNA as template directly in the PCR reaction. This is preferable as there will be some DNA coupled to the beads that will not amplify and I can remove this contamination using the magnetic properties of the beads.

 

So my question is which method is preferable. What kind of conditions would I need to elute a 500bp sequence from the beads? I'm guessing deinonized H20 at 70-92 C?

 

If I was to perform PCR directly on the beads would I want to include anything to keep the beads suspended for longer? E.g. Poly ethylene glycol? would that interfere with PCR? I know that high temperatures for PCR (i.e. 94C) will eventually destroy my beads by causing them to irreversibly aggragate, however I really only need two cycles of amplification for my purposes so this shouldn't be a problem.

 

Anyways If anyone has any idea's on this, or knows of any papers that would interest me I'd be really happy to hear of it.

[CharonY]

A former colleague of mine is actually doing PCR on beads (for 454).

The PCR is done in an emulsion. Maybe this paper helps.

 

PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets

Takaaki Kojima, Yoshiaki Takei, Miharu Ohtsuka, Yasuaki Kawarasaki, Tsuneo Yamane and Hideo Nakano*

 

Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

Nucleic Acids Research 2005 33(17):e150; doi:10.1093/nar/gni143

[Bluenoise]

Hey great thanks that is what I'm looking for. I've found tonnes of papers dealing with sequencing from beads, however unfortunatley there are some minor technical differences that come to mind between the two.

 

 

*Edit*

 

Hmmm actually it was not exactley what I was looking for. But it might serve as a starting point. The once instance that this is not carried out in emulsion uses a very small dilution of beads, so settling of the beads insn't a problem...