Methods and devices related to amplifying nucleic acid at a variety of temperatures

Abstract

Disclosed are compositions and methods for nucleic acid amplification and detection. Specifically, disclosed herein are compositions and methods that allow for amplification of nucleic acids at a wide variety of temperatures. This includes a polymerase which is thermostable at high temperatures, and a method of amplification that can be conducted at relatively low temperatures.

1 - 44 . (canceled) 
     
     
         45 . A method of amplifying a nucleic acid, the method comprising exposing a target nucleic acid to a buffer solution comprising at least four primers, wherein at least one of the four primers comprises a phosphorothioated nucleotide; and amplifying the target nucleic acid using an isothermal amplification reaction, wherein the isothermal amplification reaction produces at least one loop product, wherein at least part of the single-stranded portion of the loop product represents the target nucleic acid. 
     
     
         46 . The method of  claim 45 , wherein the loop product is exposed to a strand displacement reporter, wherein the strand displacement reporter comprises single-stranded and double-stranded nucleic acid, and further wherein a portion of the single-stranded nucleic acid of the strand displacement reporter is complementary to at least a portion of the single-stranded nucleic acid of the loop product representing the target nucleic acid, and allowing the loop product and the strand displacement reporter to interact, wherein interaction between the strand displacement reporter and the target nucleic acid portion of the loop product produces a detectable signal, wherein the signal indicates the presence of the target nucleic acid. 
     
     
         47 . The method of  claim 45 , wherein the isothermal amplification reaction is loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), polymerase spiral reaction (PSR), or helicase dependent amplification (HDA). 
     
     
         48 . (canceled) 
     
     
         49 . (canceled) 
     
     
         50 . (canceled) 
     
     
         51 . The method of  claim 45 , wherein the buffer comprises denaturant. 
     
     
         52 . The method of  claim 45 , wherein the denaturant comprises urea. 
     
     
         53 . The method of  claim 45 , wherein the buffer comprises a DNA polymerase. 
     
     
         54 . The method of  claim 45 , wherein the buffer comprises a reverse transcriptase. 
     
     
         55 . (canceled) 
     
     
         56 . (canceled) 
     
     
         57 . (canceled) 
     
     
         58 . (canceled) 
     
     
         59 . The method of  claim 45 , wherein the buffer comprises MgSO 4 . 
     
     
         60 . (canceled) 
     
     
         61 . (canceled) 
     
     
         62 . The method of  claim 45 , wherein the buffer comprises a Single-Stranded Binding (SSB) protein. 
     
     
         63 . (canceled) 
     
     
         64 . (canceled) 
     
     
         65 . The method of  claim 45 , wherein the strand displacement reporter is one step toehold displacement (OSD) reporter. 
     
     
         66 . The method of  claim 45 , wherein the target nucleic acid is RNA or DNA. 
     
     
         67 . The method of  claim 45 , wherein four primers are used with the isothermal amplification reaction. 
     
     
         68 . The method of  claim 45 , wherein five primers are used with the isothermal amplification reaction. 
     
     
         69 . The method of  claim 45 , wherein six primers are used with the isothermal amplification reaction. 
     
     
         70 . The method of  claim 45 , wherein the method comprises using at least one forward inner primer (FIP), at least one backward inner primer (BIP), at least one forward outer primer (FOP), and at least one backward outer primer (BOP). 
     
     
         71 . The method of  claim 70 , wherein at least one FIP comprises at least one phosphorothioated nucleotide. 
     
     
         72 . The method of  claim 70 , wherein at least one BIP comprises at least one phosphorothioated nucleotide. 
     
     
         73 . The method of  claim 45 , wherein two or more nucleotides of at least one primer are phosphorthioated. 
     
     
         74 . The method of  claim 45 , wherein the one or more phosphorothioated nucleotides are at the 5′-end of the primer. 
     
     
         75 . The method of  claim 45 , wherein the one or more phosphorothioated nucleotides are at the 3′-end of the primer. 
     
     
         76 - 96 . (canceled) 
     
     
         97 . The method of  claim 45 , wherein the target nucleic acid is DNA. 
     
     
         98 . The method of  claim 45 , wherein the target nucleic acid is RNA. 
     
     
         99 . The method of  claim 45 , wherein the isothermal amplification is Loop-Mediated Isothermal Amplification (LAMP). 
     
     
         100 . The method of  claim 99 , wherein LAMP is Phosphorothioated LAMP (PS-LAMP). 
     
     
         101 . The method of  claim 99 , wherein LAMP is Reverse Transcriptase LAMP (RT-LAMP).