Molecular Inversion Probes Debugging

[Mega [Andreas Stuermer]]

Hi guys!

For the lab exercise we designed molecular inversion probes. The first batch we designed didn't work, so we ordered a probe from the original paper and it still didn't work...
The main mechanism is the probe is circularized if it fits to the template.

The single stranded probe is 115 bases long. How can we check if circularization worked without radioactive labeling or expensive stuff?

I was thinking of running a gel - circular 115 bases should run slower than linear 115 bases. Though the concentration is very very low - attomols - so no chance to see on a gel.
Also, just using one primer and making multiple tandem copies (to get 230, 345, 460, ... bases) and subsequent amplification won't work (Polymerase won't kick away the primer)...

Does anyone know an easy way?

[Nathan McCorkle]

On Thu, Mar 12, 2015 at 2:06 AM, Mega [Andreas Stuermer]
<masters...@gmail.com> wrote:
> Hi guys!
>
> For the lab exercise we designed molecular inversion probes. The first batch
> we designed didn't work, so we ordered a probe from the original paper and
> it still didn't work...
> The main mechanism is the probe is circularized if it fits to the template.
>
> The single stranded probe is 115 bases long. How can we check if
> circularization worked without radioactive labeling or expensive stuff?
>
> I was thinking of running a gel - circular 115 bases should run slower than
> linear 115 bases. Though the concentration is very very low - attomols - so
> no chance to see on a gel.

Maybe evaporate some buffer to increase concentration, then introduce
to HPLC or capillary electrophoresis. If you have an old DNA sequence
available (sanger) you could probably use that for running your
sample, as long as the transfer losses aren't too great (from sample
vial, sucked into syringe/needle, then into the column).

> Also, just using one primer and making multiple tandem copies (to get 230,
> 345, 460, ... bases) and subsequent amplification won't work (Polymerase
> won't kick away the primer)...

Not sure what you mean, why would the polymerase prevent melting of the primer?


If the DNA was nicked... I wonder if you could ligate on some bigger
sequence that would stick more to a spin-column, and not elute as
quickly on the circular stuff.

[Mega [Andreas Stuermer]]

Thanks!

Not sure what you mean, why would the polymerase prevent melting of the primer?

( http://cronodon.com/images/Rolling_circle.jpg )


I meant the polymerase starts at the primer, then writes ~100 bp and then can't just read the entire DNA again five times... Like doing a spiral of the circular DNA, reading it over and ovver so you get a much longer linear fragemt from it...

[Nathan McCorkle]

I meant the polymerase starts at the primer, then writes ~100 bp and then can't just read the entire DNA again five times... Like doing a spiral of the circular DNA, reading it over and ovver so you get a much longer linear fragemt from it...

Ahh, I know there are enzymes that 'get the DNA in front out of the way'... a few terms that come to mind (and I feel like I'm forgetting the important one, but maybe not):

processivity, helicase, replication fork, replisome

Here are some links, I only scanned them, but I think they could be useful if not point you to more keywords:

http://en.wikipedia.org/wiki/Multiple_displacement_amplification

http://en.wikipedia.org/wiki/%CE%A629_DNA_polymerase

An interaction between DNA polymerase and helicase is essential for the high processivity of the bacteriophage T7 replisome.

http://www.ncbi.nlm.nih.gov/pubmed/22977246

Dynamic DNA helicase-DNA polymerase interactions assure processive replication fork movement.

http://www.ncbi.nlm.nih.gov/pubmed/17707227

Real-time single-molecule observation of rolling-circle DNA replication

http://nar.oxfordjournals.org/content/37/4/e27.full.pdf

http://en.wikipedia.org/wiki/Helicase

http://en.wikipedia.org/wiki/Replisome

last search terms I used were: 'polymerase helicase processivity rolling circle'

[Mega [Andreas Stuermer]]

Alright, thanks a lot!!!