Hybrid Multi-Step Nucleic Acid Amplification

Abstract

Improved methods for amplifying target nucleic acid sequences are provided by 1) first amplifying the number of copies of target nucleic acid sequences in a sample by a first nucleic acid amplification method, and then 2) applying a second nucleic amplification method to the amplified sample, or aliquot thereof, further amplifying the number of copies of target sequences. In embodiments, a first nucleic acid amplification method is a thermocycling method, and a second nucleic acid amplification method is an isothermal method.

1 . A method for amplifying a target nucleic acid sequence in a sample, comprising:
 first amplifying the number of copies of one or more target nucleic acid sequences in the sample by a first nucleic acid amplification method; and   next further amplifying the number of copies of said one or more target nucleic acid sequences in the sample, or an aliquot thereof, by a second nucleic amplification method.   
     
     
         2 . The method of  claim 1 , wherein said first nucleic acid amplification method comprises a thermocycling nucleic acid amplification method. 
     
     
         3 . The method of  claim 1 , wherein said second nucleic acid amplification method comprises an isothermal nucleic amplification method. 
     
     
         4 . The method of  claim 1 , wherein said first nucleic acid amplification method comprises a thermocycling nucleic acid amplification method, and said second nucleic acid amplification method comprises an isothermal nucleic amplification method. 
     
     
         5 . The method of  claim 1 , wherein said first nucleic acid amplification method comprises a polymerase chain reaction (PCR) nucleic acid amplification method. 
     
     
         6 . The method of  claim 1 , wherein said second nucleic acid amplification method comprises an isothermal nucleic amplification. 
     
     
         7 . The method of  claim 5 , wherein the nucleic acid amplified by the PCR amplification method comprises DNA. 
     
     
         8 . The method of  claim 5 , wherein the nucleic acid amplified by the PCR amplification method comprises RNA. 
     
     
         9 . The method of  claim 1 , wherein primers used in the amplification methods are directed to a single target nucleic acid sequence, and its complement. 
     
     
         10 . The method of  claim 1 , wherein primers used in the amplification methods are directed to a plurality of target nucleic acid sequences, and complements thereof. 
     
     
         11 . A method for detecting a first genetic element and a second genetic element on a common nucleic acid molecule, the method comprising:
 performing a first nucleic acid amplification reaction using a first primer and a second primer, wherein both the first primer and the second primer are phosphorylated on the 5′ end of the primer, wherein the first primer is complementary to the first genetic element, wherein the second primer is complementary to the second genetic element, and wherein a first reaction product is formed, wherein the first reaction product contains at least a portion of the first genetic element and at least a portion of the second genetic element, wherein the least a portion of the first genetic element and at least a portion of the second genetic element are separated from each other in the first reaction product by X number of nucleotides;   incubating the first reaction product with a ligase enzyme, to form at least a first ligation product containing the first reaction product, wherein within the first ligation product a copy of the at least a portion of the first genetic element and a copy of the at least a portion of the second genetic element are separated from each other by less than X number of nucleotides;   performing a second nucleic acid amplification reaction using a third primer and a fourth primer, wherein the third primer is complementary to the first genetic element, and wherein the fourth primer is complementary to the second genetic element, wherein a second reaction product is formed; and   detecting the second reaction product.   
     
     
         12 . A method for amplifying a polynucleotide template, the method comprising:
 A) generating multiple copies of a polynucleotide template in a polymerase chain reaction (PCR) amplification reaction mixture, wherein the PCR amplification reaction mixture comprises a first PCR amplification reaction primer and a second PCR amplification reaction primer, wherein in the PCR amplification reaction mixture, the first PCR amplification reaction primer anneals to the polynucleotide template and the second PCR amplification reaction primer anneals to a polynucleotide which is complementary to the polynucleotide template, and wherein in the PCR amplification reaction mixture, multiple copies of a PCR amplification reaction product are formed, wherein the PCR amplification reaction product is a double-stranded nucleic acid molecule comprising a first strand and a second strand, and wherein a first strand of the PCR amplification reaction product is a copy of the polynucleotide template;   B) incubating copies of the polynucleotide template in a non-thermocycling reaction mixture comprising a non-thermocycling reaction first primer and a non-thermocycling reaction second primer, wherein:   the polynucleotide template comprises a first portion, a second portion and a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion;   the first primer comprises a first region and a second region, wherein the second region of the first primer is complementary to the first portion of the polynucleotide template; and   the second primer comprises a first region and a second region, wherein the second region of the second primer is complementary to a sequence in the PCR amplification reaction product second strand which is complementary to the second portion of the polynucleotide template, the first region of the second primer is complementary to the first region of the first primer, and the first region of the second primer is complementary to the third portion of the polynucleotide template.   
     
     
         13 . The method of  claim 12 , wherein the first portion and second portion of the polynucleotide template are each between 6 and 30 nucleotides in length. 
     
     
         14 . The method of  claim 12 , wherein the third portion of the polynucleotide template is between 4 and 14 nucleotides in length. 
     
     
         15 . The method of  claim 12 , wherein the number of copies of the polynucleotide template in the non-thermocycling reaction mixture is increased at least 10-fold within 60 minutes of initiation of the method. 
     
     
         16 . The method of  claim 12 , wherein a concatemer strand comprising at least three copies of the polynucleotide template is generated during the incubation of the non-thermocycling reaction mixture. 
     
     
         17 . A vessel, comprising therein any one or more components of a reaction mixture provided in  claim 1 . 
     
     
         18 . A kit, comprising therein any one or more components of a reaction mixture provided in  claim 1 .