Reducing template independent primer extension and threshold time for loop mediated isothermal amplification
View Patent ↗Compositions and methods are provided for loop mediated isothermal amplification in which single stranded binding proteins are shown to protect primers from non-specific extension and to stimulate the rate of threshold amplification.
1. A method for increasing the rate of a loop mediated isothermal amplification (LAMP) reaction, comprising:
(a) combining a single-stranded binding protein (SSBP), a thermostable polymerase, at least four primers, dNTPs and a template nucleic acid in a reaction buffer at a first temperature wherein the first temperature is greater than 4° C. and less than 50° C.; and
(b) amplifying the template nucleic acid by LAMP at a second temperature that is greater than the first temperature, wherein the presence of the SSBP decreases non-templated primer extension and increases the rate of the LAMP reaction.
2. The method of claim 1 , wherein the SSBP is thermostable.
3. The method of claim 1 , wherein the thermostable polymerase is Bst DNA polymerase large fragment or a mutant thereof.
4. The method of claim 1 , wherein the at least four primers is six primers.
5. The method of claim 1 , wherein the second temperature in the range of 50° C. to 70° C.
6. The method of claim 1 , wherein the method comprises incubating the product of step (a) at a temperature of greater than 4° C. and less than 50° C. for a period of time in the range of 5 minutes to 24 hours, prior to step (b).