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Chimeric RNA/DNA oligos for 5'RLM-RACE

Chimeric RNA/DNA oligos for 5'RLM-RACE - cDNA Forum

Chimeric RNA/DNA oligos for 5'RLM-RACE - cDNA Forum. Discuss complementary DNA synthesis, cDNA libraries, and cDNA cloning in this forum.


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Old 11-05-2009, 03:17 PM
Pipette Filler
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Default Chimeric RNA/DNA oligos for 5'RLM-RACE



Part of my research involves amplification of some rather large cDNAs (6-7kb) in several different plant species. If you are familiar with the 5’ RLM RACE kit from Ambion and I was hoping you may be able to help me with a technical question.

I’ve decided to use the RLM RACE kit from Ambion, but since we already have a lot of the components, I’m ordering the primers and reagents required for the protocol. The kit uses a 5’ RACE Adapter RNA primer which is ligated to the de-capped mRNA. I’m wondering if I might be able to avoid ordering a terribly expensive primer (RNA oligo) by just specifying a conversion of the 3’ nucleoside to an RNA to enable the ligation (with the rest of the nucleosides being DNA). I thought the RT enzyme would not reverse transcribe the RNA-DNA hybrid at the cap. Do you think this approach would work?

The 5’ adapter primer sequence is 5’- GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA-3’

I would very much appreciate any thoughts or suggestions you have on this issue.

Thanks,

Jason
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Old 02-14-2011, 02:47 AM
Pipette Filler
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Default Re: Chimeric RNA/DNA oligos for 5'RLM-RACE

I think the RT enzyme such as m-mlv can reverse transcribe the RNA-DNA hybrid. You can see the article titled "Identĩcation of Novel Genes Coding for Small Expressed RNAs", in which the author used the RNA-DNA linker to the miRNA and successfully performed reverse transcription.
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5rlmrace , chimeric , oligos , rna or dna


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