SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
RNA-Seq: Orientation and expression of methicillin-resistant Staphylococcus aureus sm Newsbot! Literature Watch 0 01-05-2011 02:00 AM
Visualizing phosphorothioate oligonucleotides Bro General 5 09-08-2010 12:46 PM
PubMed: The GNUMAP Algorithm: Unbiased Probabilistic Mapping of Oligonucleotides from Newsbot! Literature Watch 0 10-29-2009 02:00 AM

Reply
 
Thread Tools
Old 10-28-2011, 08:38 AM   #1
memeri
Junior Member
 
Location: atlanta

Join Date: May 2011
Posts: 9
Default Fully exonuclease-resistant custom oligonucleotides?

A phosphorothioate linkage between the last two nucleotides makes an oligonucleotide resistant to 3' exonucleases. The phosphorothioate bond is chiral, with two stereoisomers: one is exonuclease resistant and the other is not. Standard synthesis methods are not stereo-specific, so half of the oligos produced are fully sensitive to exonuclease degradation. Illumina removes these by exposing their oligos to exonuclease and repurifying by HPLC. The resulting oligos are 100% exonuclease resistant. I don't have access to an HPLC, so my question: does anyone know of a commercial supplier of custom oligos that will provide exonuclease-pretreated (or chirally synthesized) phosphorothioate oligos?

Thanks,
Mark
memeri is offline   Reply With Quote
Old 10-28-2011, 12:01 PM   #2
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Default

You could use non-standard synthesis http://onlinelibrary.wiley.com/doi/1....nc0434s36/pdf...

I thought I had read that phosphorothioate linkages, while resistant to many exonucleases, were not resistant to T4 polymerase. If so, then the phophorothioate linkage will be of limited utility. After "end polishing + A-tailing" there is an Ampure clean up. This will likely remove the majority of exonucleases contaminating the original DNA prep. If any nuclease were to remain, one would think it would be the one that is deliberately added during "polishing" -- T4 polymerase. Or is T4 poly not used to remove 3' overhangs in the TruSeq kit?

--
Phillip
pmiguel is offline   Reply With Quote
Old 10-28-2011, 12:26 PM   #3
memeri
Junior Member
 
Location: atlanta

Join Date: May 2011
Posts: 9
Default

Thanks Phillip,

I won't be doing that synthesis! (but thanks). There are two exonucleases that cause problems. One comes along with the ligase in the adapter ligation step (I don't know if it's intrinsic to the ligase itself or just a contaminant). So the 3' T-tail on the duplex end of the adapter is linked with phosphorothioate, to prevent adapter dimer formation (perhaps less of a problem for mate pair libs that are cleaned up with streptavidin beads (the free adapter get washed away). The second exonuclease is part of the proofreading activity of the Finnzymes Phusion polymerase used in library amplification. I can't avoid that one either. So my PCR primers have a 3'-terminal phosphorothioate linkage. These primers do get chewed during PCR and it is causing some problems. Thanks again.
memeri is offline   Reply With Quote
Old 10-28-2011, 07:59 PM   #4
griffes
Junior Member
 
Location: Toronto, Canada

Join Date: Oct 2011
Posts: 4
Default

hi memri, are you sure they are treating with exonuclease? sounds like an unnecessary extra step ?
griffes is offline   Reply With Quote
Old 10-29-2011, 08:52 AM   #5
memeri
Junior Member
 
Location: atlanta

Join Date: May 2011
Posts: 9
Default

For the PCR, yes. For the ligation, probably not.
memeri is offline   Reply With Quote
Old 10-31-2011, 07:43 AM   #6
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Default

Yow! Are you talking about the PPC (PCR Primer Cocktail) from the TruSeq kits? I don't know what is going on with them. Take a look at this. They run at 80-85 nt! Makes no sense...

--
Phillip
pmiguel is offline   Reply With Quote
Reply

Tags
custom adapter primer

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 02:36 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO