Patent ID: 6531300
Filing Date: 2003-03-11
Classification: C12N,C12Q

Abstract:
A logarithmic isothermal amplification method of copying a nucleic acid sequence comprising the steps of:a. providing an aqueous solution comprising i. a target nucleic acid for amplification said target comprising a double stranded DNA having a sense strand with a first 5â€² end which bears a phage-encoded RNA polymerase recognition site and a first 3â€² end, and further having an antisense strand with a second 3â€² end and a second 5â€² end which bears a phage-encoded RNA polymerase recognition sequence, ii. a first and second amplification primer each having a phage-encoded RNA polymerase recognition sequence wherein the first primer is complementary to the second 3â€² end of the target sequence and the second primer is complementary to the first 3â€² end of the target sequence, iii. phage-encoded RNA polymerase mutated to recognize and polymerize dNTP and, iv. an excess of dNTP; b. repetitively allowing the polymerase to bind to its recognition site and to transcribe a first, short (âˆ’) copy strand of the target nucleic acid, yielding multiple copies of a primeness single (+) strand amplification product; c. creating a first amplification duplex by allowing the second primer to bind to the primeness single (+) strand amplification products of step b and permitting the polymerase to (i) extend the primer to yield a polymerase primed (âˆ’) amplification product and (ii) extend the primeness (+) strand to include a polymerase primer complement sequence creating a polymerase recognition site; d. repetitively allowing the polymerase to bind to its recognition site on the first amplification duplex and to transcribe multiple copies of a primerless single stranded (âˆ’) amplification product; e. creating a second amplification duplex by allowing primer 1 to bind to the primerless single stranded (âˆ’) amplification products of step c and permitting the polymerase (i) to extend primer 1 to yield a polymerase primed (+) amplification product and (ii) to extend the primerless (âˆ’) strand to include a polymerase primer complement sequence creating a polymerase recognition site; and, f. repetitively allowing the polymerase to bind to its recognition site on the second amplification duplex and to transcribe multiple copies of a primeness single stranded (+) amplification product.