| 摘要 |
This invention covers processes for the isothermal amplification of DNA molecules having a preselected sequence. It is based on the unexpected discovery that primers having, at some positions, adenine substituted by 2-aminopurine or diaminopurine, guanine by inosine, thymine by 2-thiothymine, and cytosine by N4-ethylcytosine (“SAMRS nucleotides”) were accepted by enzymes used in the standard helicase-dependent amplification (HDA). Further unexpected was the discovery that target nucleotides are efficiently amplified in an HDA-like process (hereinafter abbreviated as simply HDA) using substituted primers. Also discovered was the diminution of spurious products through the use of SAMRS-substituted primers. |
| 主权项 |
1. A process for synthesizing a preselected target DNA molecule, where said target DNA molecule binds to a complementary DNA molecule to form a duplex, said process comprising contacting said duplex in buffered aqueous solution with a helicase, a DNA polymerase, a single strand binding protein, 2′-deoxynucleoside triphosphates, and a substituted primer, said substituted primer that is complementary in sequence to a segment within said target DNA molecule, and where within said substituted primer one adenine in at least one of its 2′-deoxyadenosine nucleotides is substituted by 2-aminopurine or diaminopurine, or one guanine in at least one of its 2′-deoxyguanosine nucleotides is substituted by inosine, or one thymine in at least one of its thymidine nucleotides is substituted by 2-thiothymine, or at least one cytosine in at least one of its 2′-deoxycytidine nucleotides is substituted N4-ethylcytosine, and wherein the total number of said substitutions is between two and six, wherein said polymerase is Bst 2.0, GspSSD, GspM, or GspM2.0. |
