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US 11,236,384 B2Granted
Methods of RNA amplification
AssigneeAsuragen, Inc.
InventorsGary J. Latham, Richard Andrew Blidner, Liangjing Chen
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Abstract
The invention relates to methods of RNA amplification, including methods for the reverse transcription of cDNA from RNA using a thermostable reverse transcriptase. In a particular aspect, the methods are capable of linear amplification of an RNA template through multiple cycles of cDNA synthesis.
Claims24
A method for improving cDNA synthesis by linear amplification of an RNA template comprising:
- (a) contacting the RNA template with a thermostable reverse transcriptase and a first primer complementary to the RNA template, and
- (b) performing linear amplification of the RNA template that includes cDNA synthesis primed by the first primer in a reaction volume comprising betaine or a betaine analog in an amount of about 0.75 to about 3.5 M;
- wherein a cycle of cDNA synthesis includes an extension temperature and a denaturation temperature, and
- wherein the denaturation temperature is about 75 degrees C. to 90 degrees C.
1 more element
A method of linear amplification of an RNA template comprising:
- (a) contacting the RNA template with a thermostable reverse transcriptase in a reaction volume containing buffer, dNTPs, betaine or a betaine analog in an amount of about 0.75 to about 3.5 M, and a first primer complementary to the RNA template;
- (b) producing a cDNA from the RNA template in the reaction volume;
- (c) denaturing the cDNA:RNA template duplex at a temperature about 75 degrees C. to 90 degrees C.;
- (d) repeating steps (a)(c), and
- (e) producing a ratio of cDNA to RNA template of 2 to 1 or greater.
2 more elements
Dependencies are inferred from the claim text.