Single primer PCR amplification of RNA

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.

What is claimed is:  
     
         1 . 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.    
     
     
         2 . A method according to  claim 1 , wherein the 3′ target RNA hybridizing sequence is random.  
     
     
         3 . A method according to  claim 1 , wherein the 3′ target RNA hybridizing sequence is nonrandom.  
     
     
         4 . A method according to  claim 1 , wherein the 3′ target RNA hybridizing sequence is nonrandom and is complementary to a predetermined sequence selected from a coding region, a poly A junction, or a poly A tail.  
     
     
         5 . A method according to  claim 1 , wherein the first and second 5′ defined amplifiable sequences are the same.  
     
     
         6 . A method according to  claim 1 , wherein: 
 the first and second 5′ defined amplifiable sequences are different, and    the method further comprises the step of functionally depleting the first primer between steps (b) and (c).    
     
     
         7 . A method according to  claim 1 , wherein: 
 the first and second 5′ defined amplifiable sequences are different,    the method further comprises the step of functionally depleting the first primer between steps (b) and (c), and    step (e) further comprises amplifying the second cDNA strand with a fourth primer comprising the second 5′ defined amplifiable sequence.    
     
     
         8 . A method according to  claim 1 , wherein: 
 the first and second 5′ defined amplifiable sequences are different,    the method further comprises the step of functionally depleting the first primer between steps (b) and (c),    step (e) further comprises amplifying the second cDNA strand with a fourth primer comprising the second 5′ defined amplifiable sequence, and    the method further comprises step: 
 (f) amplifying the amplified cDNA with an excess of either the third or fourth primer to form a predominantly single stranded amplified probe of a predetermined orientation.  
   
     
     
         9 . A method according to  claim 1 , wherein the method is practiced in a single tube (homogeneous).  
     
     
         10 . A method according to  claim 1 , wherein the method is practiced in a single tube and the third primer of step (e) is added in functional excess of the first primer.  
     
     
         11 . A method according to  claim 1 , wherein the method comprises the step of functionally depleting remaining first and second primers between steps (d) and (e).  
     
     
         12 . A method according to  claim 1 , wherein: 
 the first and second 5′ defined amplifiable sequences are the same and    the 3′ target RNA hybridizing sequence is random.    
     
     
         13 . A method according to  claim 1 , wherein: 
 the first and second 5′ defined amplifiable sequences are the same and    the 3′ target RNA hybridizing sequence is nonrandom.    
     
     
         14 . A method according to  claim 1 , wherein: 
 the first and second 5′ defined amplifiable sequences are the same,    the 3′ target RNA hybridizing sequence is nonrandom and    the method is practiced in a single tube.    
     
     
         15 . A method according to  claim 1 , wherein: 
 the 3′ target RNA hybridizing sequence is nonrandom and    the first and second 5′ defined amplifiable sequences are different and the method further comprises the step of functionally depleting the first primer between steps (b) and (c).    
     
     
         16 . A method according to  claim 1 , wherein: 
 the 3′ target RNA hybridizing sequence is nonrandom,    the first and second 5′ defined amplifiable sequences are different and the method further comprises the step of functionally depleting the first primer between steps (b) and (c) and    the method is practiced in a single tube.    
     
     
         17 . 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.    
     
     
         18 . A method according to  claim 17 , wherein step (e) further comprises amplifying the second cDNA strand with a fourth primer comprising the second 5′ defined amplifiable sequence.  
     
     
         19 . A method according to  claim 17 , wherein step (e) further comprises amplifying the second cDNA strand with a fourth primer comprising the second 5′ defined amplifiable sequence and the method further comprises step: 
 (f) amplifying the amplified cDNA with an excess of either the third or fourth primer to form a predominantly single stranded amplified probe of a predetermined orientation.  
 
     
     
         20 . A method according to  claim 17 , wherein step (e) further comprises amplifying the second cDNA strand with a fourth primer comprising the second 5′ defined amplifiable sequence and the method further comprises step: 
 (f) amplifying the amplified cDNA with an excess of either the third or fourth primer to form a predominantly single stranded amplified probe of a predetermined orientation, and  
 the method is practiced in a single tube.  
 
     
     
         21 . 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.