Methods and systems to functionally ablate 3 prime rna ends
AssigneesSeattle Children’s Hospital d/b/a Seattle Children’s Research Institute, The Scripps Research Institute
InventorsChristian Matias Gallardo Garcia Freire, Bruce E. Torbett
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Abstract
Methods and systems to functionally ablate 3′ end of RNA are described. The functional ablation renders polymerases unable to initiate reverse transcription in the absence of an annealing primer. The methods and systems can be used to enhance the specificity and selectivity of cDNA generation from RNA.
Claims70
A method comprising:
- incubating an RNA sample with sodium periodate in a buffered solution at room temperature for 30 minutes in dark conditions, wherein the incubating results in cleavage of carbon-carbon bonds between vicinal 2′/3′ diols of the 3′ end of the RNA, converting the 2′/3′ hydroxyls into aldehydes, thereby creating 3′ ablated RNA;
- and
- incubating the 3′ ablated RNA with an annealing primer and a reverse transcriptase (RT) to generate cDNA transcribed from the 3′ ablated RNA.
A method of preparing an RNA sample for cDNA generation comprising:
- functionally-ablating the 3′ end of RNA within the RNA sample to render the functionally-ablated RNA non-transcribable by a polymerase in the absence of an annealing primer.
A method of improving cDNA yield, providing higher coverage per captured cDNA transcript, providing higher efficiency of capture of cDNA transcript with sequence lengths, reducing intergenic reads and/or reducing off-target cDNA generation thus increasing specificity of reverse transcription, the method comprising:
- Functionally-ablating the 3′ end of RNA to render the functionally-ablated RNA non-transcribable in the absence of an annealing primer, and incubating the functionally-ablated RNA with an annealing primer and a reverse transcriptase (RT) to generate cDNA transcribed from the functionally-ablated RNA, wherein the resulting cDNA has improved cDNA yield, higher coverage per captured cDNA transcript, reduced intergenic reads, and/or reduced off-target cDNA generation increasing specificity of reverse transcription, each as compared to a control cDNA generation method without the functional ablation.
Dependencies are inferred from the claim text.