View Single Post
Old 11-03-2009, 10:08 AM   #13
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,315
Default

Quote:
Originally Posted by Emanuel Heitlinger View Post
Hi all,
The SMART kit produced strange concatenated primer/adaptor artefacts.
I finally figured out what must be the source of adaptor concatamers that plague some SMART libraries.

If you are using the reverse transcriptase to add the 5' adaptor via MMLVs natural TdT activity (one to five C's are said to be added), then 5' concatamers would be the expected result. This is because MMLV will reverse transcribe RNA until it hits the end of the template, then it adds some C's and waits around for something to anneal at those C's. The 5' SMART oligo can anneal there. If it does, MMLV continues until it reaches the end of the 5' SMART oligo. (In essence MMLV has "switched" templates -- sometimes this is called "strand switching" or "template switching".)

After reaching the end of the new template (the SMART oligo) MMLV would again add non-templated C's.

This allows another 5' SMART oligo to anneal to the nascent strand, and the process can repeat over and over again, creating a string of concatamers.

The way to avoid this is to not add the 5' SMART adaptor to the reverse transcription. Instead just let the 5' SMART adaptor get added during 2nd strand synthesis/ amplification PCR. Basically a step-out from the non-templated C's that would terminate all the 1st strand cDNAs produced by MMLV. Taq polymerase doesn't add non-templated C's to the end of nascent strands, so no concatamers will result.

--
Phillip
pmiguel is offline   Reply With Quote