US2010159559A1PendingUtilityA1

Global amplification using random priming by a composite primer

76
Assignee: KURN NURITHPriority: Apr 14, 2003Filed: Oct 30, 2009Published: Jun 24, 2010
Est. expiryApr 14, 2023(expired)· nominal 20-yr term from priority
Inventors:Nurith KurnShenglong Wang
C12Q 1/6844C12Q 1/6853C12P 19/34
76
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Claims

Abstract

The invention relates to the field of polynucleotide amplification. More particularly, the invention provides methods, compositions and kits for amplification of (i.e., making multiple copies of) a multiplicity of different polynucleotide template sequences using a randomly primed RNA/DNA composite primer.

Claims

exact text as granted — not AI-modified
1 - 190 . (canceled) 
     
     
         191 . A kit comprising:
 (a) a first composite primer comprising a 5′ segment and a 3′ segment, wherein the 5′ segment comprises RNA and the 3′ segment comprises DNA, wherein said composite primer is designed to randomly hybridize to a multiplicity of sites on a template polynucleotide; and   (b) an auxiliary primer comprising DNA, wherein said auxiliary primer is designed to randomly hybridize to a multiplicity of sites on said template polynucleotide.   
     
     
         192 . A kit comprising:
 (a) a first composite primer comprising a 5′ segment and a 3′ segment, wherein the 5′ segment comprises RNA and the 3′ segment comprises DNA, wherein said composite primer is designed to randomly hybridize to a multiplicity of sites on a template polynucleotide comprising DNA; and   (b) an amplification primer, wherein said amplification primer is a composite primer comprising an RNA portion and a 3′ DNA portion, wherein the amplification primer comprises some of the sequence of the first primer, and wherein the first primer and the amplification primer are different primers.   
     
     
         193 . The kit of  claim 191  or  192 , wherein the kit further comprises a DNA dependent DNA polymerase. 
     
     
         194 . The kit of  claim 193 , wherein the DNA dependent DNA polymerase comprises strand displacement activity. 
     
     
         195 . The kit of  claim 191  or  192 , wherein the kit further comprises an RNA dependent DNA polymerase. 
     
     
         196 . The kit of  claim 191  or  192 , wherein the kit further comprises a polymerase comprising a DNA dependent DNA polymerase and an RNA dependent DNA polymerase. 
     
     
         197 . The kit of  claim 196 , wherein the kit comprises Bst polymerase. 
     
     
         198 . The kit of  claim 191  or  192 , wherein the composite primer comprises a 3′ random sequence. 
     
     
         199 . The kit of  claim 191  or  192 , wherein the composite primer is a tailed primer that comprises a 5′ portion that is not hybridizable to the template DNA under conditions in which the first primer hybridizes to the template DNA. 
     
     
         200 . The kit of  claim 191  or  192 , where the kit further comprises an agent that cleaves RNA from an RNA/DNA heteroduplex. 
     
     
         201 . The kit of  claim 200 , wherein the agent that cleaves RNA from an RNA/DNA heteroduplex is an enzyme. 
     
     
         202 . The kit of  claim 201 , wherein the enzyme is RNase H. 
     
     
         203 . The kit of  claim 202 , wherein the kit comprises an RNA dependent DNA polymerase and wherein said RNA-dependent DNA polymerase and said enzyme that cleaves RNA from an RNA/DNA heteroduplex are the same enzyme. 
     
     
         204 . The kit of  claim 202 , wherein the kit further comprises an RNA dependent DNA polymerase, and wherein said RNA dependent DNA polymerase and said enzyme that cleaves RNA from an RNA/DNA heteroduplex are different enzymes. 
     
     
         205 . The kit of  claim 202 , wherein the kit comprises a DNA dependent DNA polymerase and wherein said DNA dependent DNA polymerase and said enzyme that cleaves RNA from an RNA/DNA heteroduplex are the same enzyme. 
     
     
         206 . The kit of  claim 202 , wherein the kit further comprises a DNA dependent DNA polymerase and wherein said DNA dependent DNA polymerase and said enzyme that cleaves RNA from an RNA/DNA heteroduplex are different enzymes. 
     
     
         207 . The kit of  claim 202 , wherein the enzyme that cleaves RNA from an RNA/DNA heteroduplex comprises RNA dependent DNA polymerase activity and DNA dependent DNA polymerase activity. 
     
     
         208 . The kit of  claim 192 , wherein the RNA portion of the amplification primer is 5′ with respect to the 3′ DNA portion. 
     
     
         209 . The kit of  claim 191 , wherein the auxiliary primer comprises a hybridizing portion comprising random nucleotides. 
     
     
         210 . The kit of  claim 209 , wherein the hybridizing portion comprises a random hexamer. 
     
     
         211 . The kit of  claim 191 , wherein the auxiliary primer consists of DNA. 
     
     
         212 . The kit of  claim 191  or  192 , wherein the first composite primer comprises a hybridizing portion comprising random nucleotides. 
     
     
         213 . The kit of  claim 212 , wherein the hybridizing portion comprises DNA. 
     
     
         214 . The kit of  claim 212 , wherein the hybridizing portion is 3′ with respect to the RNA segment. 
     
     
         215 . The kit of  claim 212 , wherein the hybridizing portion comprises a random hexamer. 
     
     
         216 . The kit of  claim 192 , wherein the RNA portion of the amplification primer consists of about 7 to about 50 nucleotides. 
     
     
         217 . The kit of  claim 216 , wherein the DNA portion of the amplification primer consists of about 5 to about 20 nucleotides. 
     
     
         218 . The kit of  claim 192 , wherein the RNA portion of the amplification primer consists of about 10 to about 50 nucleotides. 
     
     
         219 . The kit of  claim 218 , wherein the DNA portion of the amplification primer consists of about 7 to about 20 nucleotides. 
     
     
         220 . The kit of  claim 191  or  192 , wherein the kit further comprises a non-canonical nucleotide. 
     
     
         221 . The kit of  claim 220 , wherein the non-canonical nucleotide is dUTP. 
     
     
         222 . The kit of  claim 191  or  192 , wherein the reaction mixture further comprises a labeled nucleotide. 
     
     
         223 . A kit comprising:
 (a) a first composite primer comprising a 5′ segment and a 3′ segment, wherein the 5′ segment comprises RNA and the 3′ segment comprises DNA, wherein said composite primer is a population of primers that are designed to hybridize to a multiplicity of sites on a template polynucleotide; and   (b) an auxiliary primer comprising DNA, wherein said auxiliary primer is designed to hybridize to a multiplicity of sites on said template polynucleotide.   
     
     
         224 . A kit comprising:
 (a) a first composite primer comprising a 5′ segment and a 3′ segment, wherein the 5′ segment comprises RNA and the 3′ segment comprises DNA, wherein said composite primer is a population of primers that are designed to hybridize to a multiplicity of sites on a template polynucleotide comprising DNA; and   (b) an amplification primer, wherein said amplification primer is a composite primer comprising an RNA portion and a 3′ DNA portion, wherein the amplification primer comprises some of the sequence of the first primer, and wherein the first primer and the amplification primer are different primers.   
     
     
         225 . The kit of  claim 223 , wherein said auxiliary primer comprise a hybridizing portion comprising random nucleotides. 
     
     
         226 . The kit of  claim 225 , wherein said auxiliary primer comprises a random hexamer. 
     
     
         227 . The kit of  claim 223 , wherein said auxiliary primer consists of DNA.

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