RNA as blocking agent in PC

pgegen at rnaworld.bio.ukans.edu pgegen at rnaworld.bio.ukans.edu
Mon Feb 12 21:47:08 EST 1996


In <n1388089521.54380 at msmtp.idde.saci.org>, brett_beitzel at MSMTP.IDDE.SACI.ORG ("Brett Beitzel") writes:
>Subject:RNA as blocking agent in PCR               Date: 2/11/96
>
>Has anyone ever tried (or ever heard of) using RNA as a blocking agent in
>PCR?
>Our lab is trying to amplify femptogram quantities of DNA with partially
>degenerate primers, so primer dimer is always a problem.  I am wondering if I
>can throw in some RNA to block primer dimer formation, while still allowing
>"legitimate" template to be amplified.
>
>Thanks for the info,
>Brett Beitzel
>brett_beitzel at msmtp.idde.saci.org
>Cancer Therapy and Research Center
>San Antonio, TX

This is unlikely to work. Any RNA from any source (yeast, bacterial, etc.)  is a mixture of fragments of cellular RNA, about 80% of which is ribosomal RNA. Since the RNA is of mixed sequence, it will undoubtedly prime in the PCR. Conceptually, any nucleic acid which could compete with primers in dimerization will also compete with primers in hybridization to the DNA template. 

It would be far better simply to design the primers so that they don't make dimers involving their 3' ends (> ca. 2 bp), or larger internal pairing (> 6 bp).

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