Patent ID: 6303306
Filing Date: 2001-10-16
Classification: C12Q

Abstract:
A method for amplifying a target nucleic acid sequence using thermostable enzymes, in which the copy number of the target nucleic acid sequence is increased in a reaction medium comprising magnesium ions and manganese ions at a ratio of about 1:1 to about 4:1 and deoxyribonucleoside and ribonucleoside triphosphates at a ratio of about 1:10 to about 10:1 at a substantially constant temperature, and where an RNA comprises a first template, that comprises the following steps:Step 1: hybridizing a first primer containing a sequence complementary to a first template RNA and a promoter sequence at the 5' terminal side thereof, with said first template RNA, which contains a target RNA optionally treated by denaturation to render it single-stranded, and elongating said first primer by thermostable RNA-dependent DNA polymerase to obtain a first primer DNA elongation product, which is a second template DNA complementary to said first template RNA;Step 2: separating said second template DNA from said first template RNA to obtain said second template DNA in single-stranded form by use of a thermostable ribonuclease H, which degrades only RNA of a DNA/RNA hybrid;Step 3: hybridizing a second primer containing a nucleic acid sequence complementary to said second template DNA with said single-stranded second template DNA and elongating said second primer by thermostable DNA-dependent DNA polymerase to obtain a second primer DNA elongation product complementary to said second template DNA, and thereby producing a double-stranded DNA intermediate containing a promoter sequence that can operate upstream of said target RNA sequence, wherein said nucleic acid sequence of said second primer is homologous to said target RNA sequence;Step 4: producing a third template RNA containing a sequence complementary to said target RNA sequence of said first template RNA from said double-stranded DNA intermediate, using thermostable DNA-dependent RNA polymerase, which can recognize said promoter sequence, wherein the thermostable DNA dependent RNA polymerase is derived from Thermus thermophilus;Step 5: hybridizing said second primer with said third template RNA in single-stranded form and elongating said second primer by use of thermostable RNA-dependent DNA polymerase to obtain said second primer DNA elongation product, which is a fourth template DNA complementary to said third template RNA;Step 6: separating said fourth template DNA from said third template RNA to obtain said fourth template DNA in single-stranded form by use of a thermostable ribonuclease H, which degrades only RNA of a DNA/RNA hybrid;Step 7: hybridizing said first primer with said single-stranded fourth template DNA and carrying out elongation by thermostable DNA-dependent DNA polymerase to obtain a first primer DNA elongation product complementary to said fourth template DNA, and a fourth template DNA elongation product complementary to said promoter sequence of said first primer, and, in such a way, a double-stranded DNA intermediate containing a promoter sequence that can operate upstream of said target RNA sequence is produced, wherein said nucleic acid sequence of said second primer is homologous to said target RNA sequence;Step 8: increasing the copy number of said third template RNA in single-stranded form containing a sequence complementary to said target RNA sequence of said first template RNA from said double-stranded DNA intermediate using thermostable DNA-dependent RNA polymerase, which can recognize said promoter sequence; andStep 9: optionally repeating said Step 5 through Step 8 as many times as required using said RNA copy, wherein a single thermostable enzyme is used as the thermostable RNA-dependent DNA Polymerase, and the thermostable DNA-dependent DNA polymerase.