Single primer PCR amplification of RNA
OwnerLyle Arnold, Erik Bjeldanes +1inventors
InventorsLyle Arnold, Erik Bjeldanes, Steve Daniel
Life of this patent
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Abstract
The invention provides methods and compositions for amplifying RNA sequences by (a) hybridizing to a target RNA a first primer comprising a 3′ target RNA hybridizing sequence and a first 5′ defined amplifyable sequence; (b) extending the first primer with a reverse transcriptase to form a first cDNA strand; (c) hybridizing to the first cDNA strand a second primer comprising a 3′ random cDNA hybridizing sequence and a second 5′ defined amplifyable sequence; (d) extending the second primer with a DNA polymerase to form a second cDNA strand; and (e) amplifying the second cDNA strand with a third primer comprising the first 5′ defined amplifyable sequence.
Claims21
A method for amplifying a RNA sequence, comprising the steps of:
- (a) hybridizing to a target RNA a first primer comprising a 3′ target RNA hybridizing sequence and a first 5′ defined amplifiable sequence;
- (b) extending the first primer with a reverse transcriptase to form a first cDNA strand;
- (c) hybridizing to the first cDNA strand a second primer comprising a 3′ random cDNA hybridizing sequence and a second 5′ defined amplifiable sequence;
- (d) extending the second primer with a DNA polymerase to form a second cDNA strand;
- and
- (e) amplifying the second cDNA strand by a polymerase chain reaction with a third primer comprising the first 5′ defined amplifiable sequence;
- wherein step (b) yields a heteroduplex of the target RNA and the first cDNA and further comprises the step of digesting the target RNA of the heteroduplex with a RNAse sufficient to permit hybridization of the first cDNA strand with the second primer without a melting step.
A method for amplifying a RNA sequence, comprising the steps of:
- (a) hybridizing to a target RNA a first primer comprising a 3′ target RNA hybridizing sequence and a first 5′ defined amplifiable sequence;
- (b) extending the first primer with a reverse transcriptase to form a first cDNA strand;
- (c) hybridizing to the first cDNA strand a second primer comprising a 3′ random cDNA hybridizing sequence and a second 5′ defined amplifiable sequence;
- (d) extending the second primer with a DNA polymerase to form a second cDNA strand;
- and
- (e) amplifying the second cDNA strand with a third primer comprising the first 5′ defined amplifiable sequence;
- wherein the 3′ target RNA hybridizing sequence is random;
- the first and second 5′ defined amplifiable sequences are different;
- and
- step (b) comprises the step of functionally depleting the first primer to prevent it from hybridizing with the first cDNA strand in subsequent steps.
A composition for amplifying a RNA sequence, said composition comprising instructions reciting the steps of:
- (a) hybridizing to a target RNA a first primer comprising a 3′ target RNA hybridizing sequence and a first 5′ defined amplifiable sequence;
- (b) extending the first primer with a reverse transcriptase to form a first cDNA strand;
- (c) hybridizing to the first cDNA strand a second primer comprising a 3′ random cDNA hybridizing sequence and a second 5′ defined amplifiable sequence;
- (d) extending the second primer with a DNA polymerase to form a second cDNA strand;
- and
- (e) amplifying the second cDNA strand by a polymerase chain reaction with a third primer comprising the first 5′ defined amplifiable sequence;
- wherein step (b) is said to yield a heteroduplex of the target RNA and the first cDNA and further comprises the step of digesting the target RNA of the heteroduplex with a RNAse sufficient to permit hybridization of the first cDNA strand with the second primer without a melting step.
Dependencies are inferred from the claim text.