Methods and compositions for linear isothermal amplification of polynucleotide sequences

Abstract

The present invention provides novel isothermal, single primer linear nucleic acid amplification methods. Methods for amplifying complementary DNA using a composite primer, primer extension, strand displacement, and optionally a termination sequence, are provided. Methods for amplifying sense RNA using a composite primer, primer extension, strand displacement, optionally template switching, a propromoter oligonucleotide and transcription are also provided. The invention further provides compositions and kits for practicing said methods, as well as methods which use the amplification products.

What is claimed is:  
     
         1 . A method for amplifying a polynucleotide sequence complementary to a target polynucleotide sequence comprising: 
 (a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3′ DNA portion;    (b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′ with respect to hybridization of the composite primer to the template;    (c) extending the composite primer with DNA polymerase;    (d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer can hybridize to the template and repeat primer extension by strand displacement,    whereby multiple copies of the complementary sequence of the target sequence are produced.    
     
     
         2 . A method for amplifying a target polynucleotide sequence comprising: 
 (a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3′ DNA portion;    (b) hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′ with respect to hybridization of the composite primer to the template;    (c) extending the composite primer with DNA polymerase;    (d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer can hybridize to the template and repeat primer extension by strand displacement to produce displaced primer extension product;    (e) hybridizing a polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions which allow transcription to occur by RNA polymerase, such that RNA transcripts are produced comprising sequences complementary to the displaced primer extension products,    whereby multiple copies of the target sequence are produced.    
     
     
         3 . The method of claims  1  or  2 , wherein the RNA portion of the composite primer is 5′ with respect to the 3′ DNA portion.  
     
     
         4 . The method of    claim 3   , wherein the 5′ RNA portion is adjacent to the 3′ DNA portion.  
     
     
         5 . The method of claims  1  or  2 , wherein a plurality of composite primers are used.  
     
     
         6 . The method of claims  1  or  2 , wherein the polynucleotide comprising a termination sequence is a template switch oliognucleotide (TSO).  
     
     
         7 . The method of    claim 6   , wherein the TSO comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the TSO binds more tightly to the region as compared to a TSO without the modification.  
     
     
         8 . The method of claims  1  or  2 , wherein the polynucleotide comprising a termination sequence is a blocking sequence.  
     
     
         9 . The method of    claim 8   , wherein the blocking sequence comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the blocking sequence binds more tightly to the region as compared to a blocking sequence without the modification.  
     
     
         10 . The method of claims  1  or  2 , wherein the enzyme that cleaves RNA is RNaseH.  
     
     
         11 . The method of    claim 2   , wherein the polynucleotide comprising a propromoter and region which hybridizes to the displaced primer extension product is a template switch oligonucleotide (TSO).  
     
     
         12 . The method of    claim 2   , wherein the polynucleotide comprising the propromoter comprises a region at the 3′ end which hybridizes to the displaced primer extension product, whereby DNA polymerase extension of displaced primer extension product produces a double stranded promoter from which transcription occurs.  
     
     
         13 . The method of    claim 12   , wherein the polynucleotide comprising the propromoter is a propromoter template oligonucleotide (PTO).  
     
     
         14 . The met hod of claims  1  or  2 , wherein steps (a) and (b) are performed in either order.  
     
     
         15 . The method of claims  1  or  2 , wherein steps (a) and (b) are performed simultaneously.  
     
     
         16 . The method of claims  1  or  2 , wherein steps (a), (b) and (c) are performed simultaneously.  
     
     
         17 . The method of claims  1  or  2 , wherein steps (a) and (b) are performed before step (c).  
     
     
         18 . The method of claims  1  or  2 , wherein all steps are performed simultaneously.  
     
     
         19 . A method of sequencing a target nucleotide sequence comprising: 
 (a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3′ DNA portion;    (b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′ with respect to hybridization of the composite primer to the template;    (c) extending the composite primer to a termination site with DNA polymerase and a mixture of dNTPs and dNTP analogs, such that primer extension is terminated upon incorporation of a dNTP analog;    (d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer can hybridize to the template and repeat primer extension by strand displacement, whereby multiple copies of the complementary sequence of the target sequence are produced of varying lengths;    (e) analyzing the product of steps (a) through (d) to determine sequence.    
     
     
         20 . A method of sequencing a target nucleotide sequence comprising 
 (a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3′ DNA portion;    (b) hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′ with respect to hybridization of the composite primer to the template;    (c) extending the composite primer with DNA polymerase;    (d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer can hybridize to the template and repeat primer extension by strand displacement to produce displaced primer extension product;    (e) hybridizing a polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions such that transcription occurs from the extension product by RNA polymerase, using a mixture of rNTPs and rNTP analogs, such that RNA transcripts are produced comprising sequences complementary to the displaced primer extension products, and such that transcription is terminated upon incorporation of an rNTP analog, whereby multiple copies of the target sequence are produced of varying lengths;    (f) analyzing the product of steps (a) through (e) to determine sequence.    
     
     
         21 . The method of    claim 19    or    20   , wherein the RNA portion of the composite primer is 5′ with respect to the 3′ DNA portion.  
     
     
         22 . The method of    claim 21   , wherein the 5′ RNA portion is adjacent to the 3′ DNA portion.  
     
     
         23 . A method of characterizing a sequence of interest in a target polynucleotide, said method comprising 
 conducting the method of claims  1  or  2  wherein the sequence of an RNA portion of the composite primer is known, and wherein    (a) production of detectably fewer amplification products from the template as compared to the amount of amplification products from a reference template which comprises a region complementary to the RNA portion of the composite primer indicates that the target polynucleotide does not comprise a sequence complementary to the RNA portion of the composite primer and is a sequence variant with respect to the sequence complementary to the RNA portion of the composite primer; or    (b) production of detectably more amplification products from the template as compared to the amount of amplification products from a reference template which does not comprise a region which is complementary to the RNA portion of the composite primer indicates that the target polynucleotide comprises a sequence complementary to the RNA portion of the composite primer and is not a sequence variant with respect to the sequence complementary to the RNA portion of the composite primer.    
     
     
         24 . The method of    claim 23   , wherein the sequence of an RNA portion of the composite primer comprises a wild type sequence, and the sequence of interest is characterized in determining the presence or absence of the wild type sequence.  
     
     
         25 . The method of    claim 23   , wherein the sequence of an RNA portion of the composite primer comprises a mutant sequence, and the sequence of interest is characterized in determining the presence or absence of the mutant sequence.  
     
     
         26 . The method of    claim 23   , wherein the sequence of an RNA portion of the composite primer comprises an allelic sequence, and the sequence of interest in characterized in determining the presence or absence of the allelic sequence.  
     
     
         27 . A method of detecting a mutation in a target polynucleotide, comprising 
 (a) conducting the method of claims  1  or  2 ; and (b) analyzing the amplified products of the method for single stranded conformation, wherein a difference in conformation as compared to a reference single stranded polynucleotide indicates a mutation in the target polynucleotide.    
     
     
         28 . The method of claims  23 , wherein the RNA portion of the composite primer is 5′ with respect to the 3′ DNA portion.  
     
     
         29 . The method of    claim 28   , wherein the 5′ RNA portion is adjacent to the 3′ DNA portion.  
     
     
         30 . A method of producing a microarray, comprising (a) conducting the amplification method of claims  1  or  2 ; and (b) attaching the amplified products onto a solid substrate to make a microarray of the amplified products.  
     
     
         31 . A composition comprising a composite primer, said composite primer comprising a 3′ DNA portion and a 5′ RNA portion.  
     
     
         32 . The composition of    claim 31   , wherein the 5′ RNA portion is adjacent to the 3′ DNA portion.  
     
     
         33 . The composition of    claim 31   , wherein the 5′ RNA portion is about 5 to about 20 nucleotides and the 3′ DNA portion is about 5 to about 15 nucleotides.  
     
     
         34 . A composition comprising a TSO, wherein the TSO comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the TSO binds more tightly to the region as compared to a TSO without the modification.  
     
     
         35 . A composition comprising the composite primer of    claim 31    and the TSO of    claim 34   .  
     
     
         36 . A composition comprising the composite primer of    claim 31    and a blocking sequence.  
     
     
         37 . A composition comprising the composite primer of    claim 31    and a propromoter template oligonucleotide (PTO).  
     
     
         38 . A composition comprising a complex of (a) a template strand; and (b) a composite primer, said composite primer comprising a 3′ DNA portion and an RNA portion.  
     
     
         39 . The composition of    claim 38   , wherein the RNA portion is 5′ and adjacent to the 3′ DNA portion.  
     
     
         40 . The composition of    claim 39   , wherein the complex further comprises a termination sequence.  
     
     
         41 . The composition of    claim 40   , wherein the termination sequence is a TSO.  
     
     
         42 . The composition of    claim 40   , wherein the termination sequence is a blocking sequence.  
     
     
         43 . A reaction mixture comprising (a) a polynucleotide template; (b) a composite primer comprising a 3′ DNA portion and an RNA portion; and (c) DNA polymerase.  
     
     
         44 . The reaction mixture of    claim 43   , wherein the composite primer comprises a 5′ RNA portion which is adjacent to the 3′ DNA portion.  
     
     
         45 . The reaction mixture of    claim 44   , further comprising an enzyme which cleaves RNA from an RNA/DNA hybrid.  
     
     
         46 . The reaction mixture of    claim 45   , wherein the enzyme is RNaseH.  
     
     
         47 . The reaction mixture of    claim 44   , further comprising a polynucleotide comprising a termination polynucleotide sequence.  
     
     
         48 . The reaction mixture of    claim 47   , further comprising a polynucleotide comprising a propromoter.  
     
     
         49 . The reaction mixture of    claim 48   , wherein the polynucleotide comprising a propromoter is a TSO.  
     
     
         50 . The reaction mixture of    claim 48   , wherein the polynucleotide comprising a propromoter is a propromoter template oligonucleotide (PTO).  
     
     
         51 . A kit for amplification of a target polynucleotide sequence, comprising a composite primer comprising a 3′ DNA portion and an RNA portion.  
     
     
         52 . The kit of    claim 51   , wherein the RNA portion is 5′ with respect to the 3′ DNA portion.  
     
     
         53 . The kit of    claim 52   , wherein the 5′ RNA portion is adjacent to the 3′ DNA portion.  
     
     
         54 . The kit of    claim 51   , further comprising a polynucleotide comprising a termination polynucleotide sequence.  
     
     
         55 . The kit of    claim 54   , wherein the termination polynucleotide sequence is a TSO.  
     
     
         56 . The kit of    claim 54   , wherein the termination polynucleotide sequence is a blocker sequence.  
     
     
         57 . The kit of    claim 51   , further comprising a polynucleotide comprising a propromoter.  
     
     
         58 . The kit of    claim 57   , wherein the polynucleotide comprising a propromoter is a TSO.  
     
     
         59 . The kit of    claim 57   , wherein the polynucleotide comprising a propromoter is a PTO.  
     
     
         60 . The kit of    claim 51   , further comprising an enzyme which cleaves RNA from an RNA/DNA hybrid.  
     
     
         61 . The kit of    claim 60   , wherein the enzyme is RNaseH.  
     
     
         62 . A system for amplifying a target polynucleotide sequence or its complement, comprising (a) a composite primer comprising a 3′ DNA portion and an RNA portion; (b) DNA polymerase; and (c) an enzyme which cleaves RNA from an RNA/DNA hybrid.  
     
     
         63 . The system of    claim 62   , wherein the enzyme is RNaseH.  
     
     
         64 . The system of claims  62  or  63 , wherein the composite primer comprises a 5′ RNA portion which is adjacent to the 3′ DNA portion.