Isothermal nucleic acid amplification methods and compositions

Abstract

Methods and compositions are provided related to the amplification of target polynucleotide sequences as well as total RNA and total DNA amplification. In some embodiments, the methods and compositions also allow for the immobilization and capture of target polynucleotides with defined 3′ and or 5′ sequences to solid surfaces. The polynucleotides attached to the solid surfaces can be amplified or eluted for downstream processing. In some cases, nucleotides attached to solid surfaces can be used for high throughput sequencing of nucleotide sequences related to target DNA or target RNA.

1 .- 67 . (canceled) 
     
     
         68 . A method of capturing and amplifying a target sequence or sequences comprising:
 (a) providing a surface wherein an all-DNA first primer or set of all-DNA first primers comprising a 5′ tail sequence comprising sequence (A) and a 3′ template annealing sequence comprising sequence (P) are immobilized on said surface;   (b) annealing a target nucleic acid to said first primer or set of primers;   (c) extending the first primer or set of primers with a DNA polymerase to produce an immobilized primer extension product or set of immobilized primer extension products complimentary to and hybridized with the target nucleic acid to form a complex or set of complexes;   (d) dissociating the target nucleic acid-primer extension product complex or set of primer extension product complexes;   (e) annealing a second primer comprising a 3′ DNA template annealing sequence (P) or set of template annealing sequences and a 5′ RNA tail sequence comprising sequence (A) or sequence (B) to the immobilized primer extension product or products, wherein sequence B is different from sequence A; and   (f) extending said second primer with a DNA polymerase to create a second primer extension product or set of primer extension products comprising a double stranded nucleic acid comprising a 3′ DNA-DNA duplex (A)-(A′), wherein A′ is the complement of A.   
     
     
         69 . The method of  claim 68  wherein said template nucleic acid is selected from the group consisting of RNA and DNA. 
     
     
         70 . The method of  claim 68  wherein said first primer comprises a spacer element between the immobilizing surface and the remaining portion of the primer, a 3′ DNA end comprising a 3′ DNA annealing sequence (P), and a 5′ comprising a common 5′ DNA sequence tail (A) or sequence tail (B) between said spacer element and annealing sequence. 
     
     
         71 . The method of  claim 68  wherein the set of first primers comprises:
 (a) a spacer element between the immobilizing surface and the remaining portion of the primer; 
 (b) a 3′ DNA end wherein members of the set of first primers comprise unique target specific 3′ DNA annealing sequences (P); and 
 (c) a 5′ DNA end comprising a common 5′ DNA sequence tail (A) or DNA sequence tail (B) between said spacer element and annealing sequence. 
 
     
     
         72 . The method of  claim 68  wherein said annealing sequences (P) of said set of first primers are each specific to a target or region of the template nucleic acid. 
     
     
         73 . The method of  claim 68  wherein the 5′ tail sequence of the first primer is sequence (A) and the 5′ tail sequence of the second primer is sequence (B). 
     
     
         74 . The method of  claim 68  wherein the 5′ tail sequence of the first primer is sequence (A) and the 5′ tail sequence of the second primer is sequence (A). 
     
     
         75 . The method of  claim 68  wherein said DNA polymerase does not exhibit strand displacement activity. 
     
     
         76 . The method of  claim 68  further comprising step (g) dissociation of the second primer extension product by heat or chemical denaturation. 
     
     
         77 . The method of  claim 68  wherein the surface is selected from the group consisting of a bead, a magnetic particle, a microarray, a gene chip, and an array. 
     
     
         78 . The method of amplifying the eluted DNA polymerase product of  claim 76  further comprising:
 (h) annealing an amplification primer comprising a 5′ RNA sequence and a 3′DNA sequence; 
 (i) extending said annealed primer with a DNA polymerase; and 
 (j) amplifying the eluted DNA polymerase product with a reaction mixture comprising RNase H, an amplification primer comprising a DNA portion and a 5′ RNA portion, and a DNA polymerase with strand displacement activity to produce an amplified DNA product or products having a defined 5′ sequence (A′) or (B′). 
 
     
     
         79 . The method of  claim 68  further comprising:
 (g) degradation of the single stranded 3′ end of said first primer extension product and single stranded immobilized first primers with a single stranded DNA specific 3′ exonuclease; 
 (h) extending the 3′ end of the first primer extension product with an RNA-dependent DNA polymerase to generate an immobilized double stranded product with an RNA-DNA heteroduplex at one end. 
 (i) cleavage of spacer element with light or chemical cleavage to release the immobilized double stranded nucleic acid from the surface; and 
 (j) amplifying the released double stranded nucleic acid in solution with a reaction mixture comprising RNase H, an amplification primer comprising a DNA portion and a 5′ RNA portion, and a DNA polymerase with strand displacement activity to produce an amplified DNA product or products having a defined 3′ sequence comprising sequence (A′). 
 
     
     
         80 . The method of  claim 68  further comprising:
 (g) degrading the single stranded 3′ end of said first primer extension product or products and unextended immobilized first primers with a single stranded DNA specific 3′ exonuclease; 
 (h) extending said first primer extension product with a DNA polymerase; and 
 (i) degrading RNA in the heteroduplex from the first primer extension product and the RNA portion of the second primer with RNAse H. 
 (j) annealing an amplification primer to the single stranded portion of the second primer extension product wherein the amplification primer has a DNA portion and a 5′ RNA portion; 
 (k) extending the amplification primer with a DNA polymerase having strand displacement activity to produce an amplified product hybridized to the second primer extension product; and 
 (l) repeating steps (i)-(k) to produce multiple copies of amplified product or products having a defined 3′ sequence comprising sequence (A′). 
 
     
     
         81 . The method of  claim 74  further comprising:
 (g) separating the second primer extension product or products from the first primer extension product or products by heat or chemical denaturation; 
 (h) annealing the second primer extension product in solution to create a stem loop structure with a DNA sequence (A′)-RNA sequence (A) heteroduplex end; 
 (i) degrading RNA in the heteroduplex with RNase H; 
 (j) annealing an amplification primer to the single stranded portion of the second primer extension product wherein the amplification primer has a DNA portion and a 5′ RNA portion, wherein the RNA portion of the amplification primer forms a heteroduplex with a DNA portion of the second primer extension product; 
 (k) extending the amplification primer with a DNA polymerase having strand displacement activity to produce an amplified product hybridized to the second primer extension product to form a double stranded nucleic acid, said amplified product forming an RNA-DNA heteroduplex at one end of the double stranded nucleic acid; and 
 (l) repeating steps (i)-(k) to produce amplified DNA. 
 
     
     
         82 . The method of  claim 81  wherein between step (f) and step (g), a single stranded DNA specific 3′ exonuclease is added to degrade unincorporated primers.