Helicase dependent amplification of DNA molecules using nucleotide analogs
OwnerSteven A Benner, Zunyi Yanginventors
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Abstract
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.
Claims8
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.
An amplification process for creating multiple copies of a target DNA molecule, 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 two substituted primers, a forward primer and a reverse primer, said forward primer binding to a segment within said target DNA molecule, and said reverse primer being substantially identical in sequence to a segment downstream within said target DNA molecule, and where within both of said substituted primers 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 by 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.
Dependencies are inferred from the claim text.