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Difference between streptavidin-bound nucleic acid probe and probe covalently at

molecular biology

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#1 FreddieP

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Posted 17 July 2013 - 06:35 AM

Hi,
Thanks for reading my question. I am not very experienced here so I would be grateful for any insight.

I want to attach a probe to a solid support and use it to bind a mRNA. In the microarray the probe is just attached to the support but other techniques use a biotinylated probe bound to bound streptavidin to capture the target. What is the difference between the binding efficiency of a strepatividn bound probe and a probe that is stuck on the support by other means?

Thanks, FreddieP

#2 bob1

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Posted 17 July 2013 - 01:38 PM

The affinity and specificity between streptavidin and biotin is extremely high (you should be able to look up binding constants and the like). However, this will pick up ANY biotinylated molecule but tells you nothing about which molecules you have. Conversely the idea behind microarray is that each specific sequence attached to the chip has a different and specific target, so that you can identify which attached sequences have bound a target mRNA, and can from that deduce patterns of gene expression.

#3 FreddieP

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Posted 18 July 2013 - 07:22 AM

I am referring to the situation when the specific probe is labelled with biotin and bound to streptavidin, which is bound to the support. Instead of the specific probe directly to the support, the probe is bound to streptavidin. Why do people use streptavidin to bind the probe when they could just attach the probe to the support?

#4 bob1

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Posted 18 July 2013 - 12:56 PM

There will be three reasons 1) - it is relatively easy (chemically speaking) to attach streptavidin to surfaces, 2) - streptavidin is abundant and easy to prepare, 3) - it is easy to biotinylate probes and therefore easy to customize what is added where.

#5 Winegarden

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Posted 19 July 2013 - 09:32 AM

Hi Freddie,

There are a few reasons to do this. The first is that you can sometimes get better kinetics with a biotin-oligo bound to an avidin surface. Using direct covalent coupling usually requires some sort of aliphatic linker (C6 or C12 liner) to help get the oligo up off of the surface in order to improve hybridisation. Using biotin-avidin can accomplish a similar effect.

Also - some of the covalent chemistries like those which use aldehyde surfaces - the reaction that occurs to create the imine bond (Schiff Base) is actually reversible. The imine needs to be reduced by something like sodium borohydride to create a more stable bond. This reduction reaction can potentially cause problems if for example you want to use a fluorphore labelled oligo. The strepatvidin-biotin of course occurs rapidly and is very tight and essentially non-reversable.

That all being said - the vast majority of DNA arrays use either eletrostatic type interactions or use covalent linkages (aldehyde or epoxy surface). Many people reported the biotin approach but it never really took off because in practice is tended to perform poorly (higher background, lower dynamic range etc...).

#6 FreddieP

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Posted 20 July 2013 - 12:24 PM

Is streptavidin easy to prepare? I hear that it is not easy to prepare although I have never done it. Also, I am interested in expense. You can't buy strepatividin cheaply anywhere. A few companies are coming out with large batches of streptavidin but mostly you only can buy small batches, very costly. The expense of purifying it yourself is unknown to me. Direct binding seems less costly but the efficiency of binding the target is also important.





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