Oscillating Amplification Reaction For Nucleic Acids

Abstract

One embodiment of the present invention provides for a method for amplifying a template of nucleic acid target sequence contained in a sample. The method includes contacting the sample with an amplification reaction mixture containing a primer complementary to the template of nucleic acid target sequence. A temperature of the reaction is oscillated between an upper temperature and a lower temperature wherein the change in temperature is no greater than about 20° C. during a plurality of temperature cycles. The template of nucleic acid target sequence is amplified.

What is claimed is: 
     
         1 . A method for amplifying a target nucleic acid sequence contained in a sample, the method comprising:
 contacting the sample with an amplification reaction mixture comprising a primer complementary to the target nucleic acid sequence;   oscillating a temperature of the reaction between an upper temperature and a lower temperature wherein a change in temperature is no greater than about 20° C. during a plurality of temperature cycles; and   amplifying the target nucleic acid sequence.   
     
     
         2 . The method of  claim 1  wherein the change In temperature is no greater than about 15° C. during a plurality of temperature cycles. 
     
     
         3 . The method of  claim 1  wherein the change in temperature is no greater than about 10° C. during a plurality of temperature cycles. 
     
     
         4 . The method of  claim 1  wherein the change in temperature is no greater than about 5° C. during a plurality of temperature cycles. 
     
     
         5 . The method of  claim 1  wherein upon reaching the upper temperature or the lower temperature, the temperature is maintained for a set period of time within a temperature fluctuation. 
     
     
         6 . The method of  claim 1  wherein upon reaching an upper or lower temperature within the temperature range, the temperature is varied to the other temperature. 
     
     
         7 . The method of  claim 1  wherein the lower temperature is no less than about 50° C. 
     
     
         8 . The method of  claim 1  wherein the upper temperature is no greater than about 85° C. 
     
     
         9 . The method of  claim 1  wherein the target nucleic acid sequence comprises single stranded DNA or RNA. 
     
     
         10 . The method of  claim 1  wherein the target nucleic acid sequence comprises double stranded DNA or RNA. 
     
     
         11 . The method of  claim 1  wherein the target nucleic acid sequence is RNA. 
     
     
         12 . The method of  claim 1  wherein the target nucleic acid sequence is DNA. 
     
     
         13 . The method of  claim 1  wherein the length of the target nucleic acid sequence is less than about 1000 bp. 
     
     
         14 . The method of  claim 1  wherein the length of the target nucleic acid sequence is less than about 250 bp. 
     
     
         15 . The method of  claim 1  wherein the length of the target nucleic acid sequence is less than about 150 bp. 
     
     
         16 . The method of  claim 1  wherein the length of the target nucleic acid sequence is be less than about 100 bp. 
     
     
         17 . The method of  claim 1  wherein the amplification reaction mixture comprises a pair of primers which bind to opposite strands of the target nucleic acid sequence. 
     
     
         18 . The method of  claim 17  wherein the each primer of the primer pair has a length and a GC content so that its melting temperature is ≥65° C. 
     
     
         19 . The method of  claim 18  wherein the each primer of the primer pair has a length and a GC content so that the melting temperature is ≥70° C. 
     
     
         20 . The method of  claim 19  wherein the melting temperature of each primer of the primer pair is between 70-80° C. 
     
     
         21 . The method of  claim 17  wherein the each primer of the primer pair has a length of 35-70 base pairs. 
     
     
         22 . The method of  claim 17  wherein the pair of primers comprise a forward primer and a reverse primer each having a length of 40-47 base pairs. 
     
     
         23 . The method of  claim 1  wherein the amplification reaction mixture comprises a nucleic acid destabilizing agent. 
     
     
         24 . The method of  claim 23  wherein the destabilizing agent is selected from the group consisting of dimethylsulfoxlde (DMSO), formamide, Betaine, and combinations thereof. 
     
     
         25 . The method of  claim 23  wherein a concentration of the nucleic acid destabilizer is from 8-15 volume percent. 
     
     
         26 . The method of  claim 23  wherein the destabilizing agent is a single strand or double strand nucleic acid destabilizing agent. 
     
     
         27 . The method of  claim 1  wherein the amplification reaction mixture comprises a DNA polymerase. 
     
     
         28 . The method of  claim 27  wherein the DNA polymerase is buffered at a pH to support activity. 
     
     
         29 . The method of  claim 27  wherein the DNA polymerase is a thermostable DNA polymerase. 
     
     
         30 . The method of  claim 27  wherein the DNA polymerase is selected from the group consisting of TAQ DNA polymerase, VentR DNA polymerase, and DeepVentR DNA polymerase. 
     
     
         31 . The method of  claim 27  wherein the DNA polymerase comprises a strand displacing activity. 
     
     
         32 . The method of  claim 27  wherein the DNA polymerase does not have 3′−>5′ exonuclease activity. 
     
     
         33 . The method of  claim 1  wherein the amplification reaction mixture comprises a reverse transcriptase. 
     
     
         34 . The method of  claim 33  wherein the reverse transcriptase is a thermostable reverse transcriptase. 
     
     
         35 . The method of  claim 33  wherein the reverse transcriptase is selected from AMV-RT, Superscript II reverse transcriptase, Superscript III reverse transcriptase, or MMLV-RT. 
     
     
         36 . The method of  claim 1  wherein the amplification reaction mixture comprises a single stranded binding protein. 
     
     
         37 . The method of  claim 36  wherein the single stranded binding protein is a thermal stable single stranded binding protein. 
     
     
         38 . The method of  claim 36  wherein the single stranded binding protein is a non-thermal stable single stranded binding protein. 
     
     
         39 . The method of  claim 1  wherein the sample is not alcohol free. 
     
     
         40 . The method of  claim 1  wherein the sample is not salt free. 
     
     
         41 . The method of  claim 1  wherein the upper temperature is a denaturation temperature and a lower temperature is an annealing temperature. 
     
     
         42 . The method of  claim 41  wherein the upper denaturation temperature is sufficiently high to completely denature the target nucleic acid sequence. 
     
     
         43 . The method of  claim 1  further comprising mixing a solution comprising a divalent cation together with other components to form the amplification reaction mixture. 
     
     
         44 . The method of  claim 43  wherein the solution is a buffer. 
     
     
         45 . The method of  claim 43  wherein the divalent cation is a salt composing an element selected from the group consisting of magnesium, manganese, copper, zinc and any combination thereof. 
     
     
         46 . The method of  claim 43  wherein the solution comprises a monovalent cation. 
     
     
         47 . The method of  claim 46  wherein the monovalent cation is a salt comprising an element selected from the group consisting of sodium, potassium, lithium, rubidium, cesium, ammonium and any combination thereof.