Isothermal strand displacement amplification using primers containing a non-regular base
Inventors
Millar, Douglas Spencer • Melki, John R. • Grigg, Geoffrey W.
Assignees
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Publication Number
US-8431347-B2
Publication Date
2013-04-30
Expiration Date
Abstract
The invention is directed to a method for isothermal DNA amplification comprising providing to the DNA to be amplified an amplification mix comprising a first primer at least partially complementary to a region of DNA and containing a non-regular base, a second primer at least partially complementary to a region of DNA and containing a non- regular base, a DNA polymerase, an enzyme capable of strand displacement, an enzyme that recognises a non-regular base in double-stranded DNA and causes a nick or excises a base in one DNA strand at or near the non-regular base; and amplifying the DNA substantially without thermal cycling.
Core Innovation
The invention relates to an isothermal DNA amplification method that uses primers and specific nucleic-acid enzymes to amplify DNA substantially without thermal cycling. A primer containing at least one deoxyinosine, and not including a ribonucleotide, is at least partially complementary to a region having a CpG dinucleotide, and inosine substitutes the position of guanine in a CpG dinucleotide of the DNA. The method uses an exonuclease deficient DNA polymerase and an enzyme capable of strand displacement, together with an enzyme that recognizes a deoxyinosine in double-stranded DNA and causes a nick in one DNA strand at or near the deoxyinosine.
Amplification proceeds via primer-template complex formation, 3′ extension with an exonuclease deficient DNA polymerase to generate a double stranded molecule with a newly synthesized strand containing inosine bases, followed by causing nicks at or near the inosine base using a nicking enzyme. After nicking, the newly synthesized strand is displaced and regenerated with a DNA polymerase having strand displacement activity, and newly synthesized strands are formed by successive rounds of nicking and strand displacement.
In addition, the method specifies that inosine substitutions are made at CpG dinucleotide positions of the DNA template by using inosine-containing primers, including cases where the first newly synthesized strand contains a plurality of inosine bases. The invention further includes embodiments that incubate the extension product with an endonuclease V to generate nicks at positions 3′ of the incorporated inosine, and then extends from the nicks with strand displacing enzymes, repeating at least once to generate amplified template DNA.
Claims Coverage
The independent claims cover multiple variants of an inosine-in-CpG, strand-displacement, nicking-based DNA amplification workflow using exonuclease deficient DNA polymerase plus a nicking enzyme that recognizes inosine in double-stranded DNA, repeated under isothermal or defined conditions. Across the independent claims, the inventive features focus on CpG-targeting primers with inosine substitution, exonuclease deficient strand-displacing polymerase extension, nick generation at or near the inosine position, and successive rounds that amplify the DNA without thermal cycling.
Inosine-substituted CpG targeting with isothermal amplification
An isothermal DNA amplification method where an amplification mix includes first and second primers at least partially complementary to a region having a CpG dinucleotide, each containing at least one deoxyinosine and not including a ribonucleotide, an exonuclease deficient DNA polymerase, an enzyme capable of strand displacement, and an enzyme that recognizes a deoxyinosine in double-stranded DNA and causes a nick at or near the deoxyinosine; inosine substitutes the position of guanine in a CpG dinucleotide of the DNA, and DNA is amplified substantially without thermal cycling.
Iterative nicking and strand-displacement amplification with inosine-containing primer
A method for amplifying DNA comprising hybridizing an inosine-containing primer to one strand at a CpG dinucleotide region, extending the 3′ end using an exonuclease deficient DNA polymerase to generate a double stranded molecule with a first newly synthesized strand containing inosine that substitutes for guanine in a CpG dinucleotide, causing a nick at or near the inosine base using a nicking enzyme, displacing the first newly synthesized strand and regenerating the inosine-containing primer with a DNA polymerase having strand displacement activity, and repeating the nicking and displacement steps to amplify DNA via successive rounds.
Double-strand generation, nicking at inosine bases, and strand-displacement copying
A method for amplifying a template DNA comprising binding a primer containing an inosine base to template DNA at regions having CpG dinucleotides, extending the primer in the presence of an exonuclease deficient DNA polymerase with strand displacement capability to generate a double stranded molecule with a first newly synthesized strand containing a plurality of inosine bases that substitute for guanine in the CpG dinucleotides, causing nicks at or near the inosine bases using a nicking enzyme, extending from the nicks with a DNA polymerase having strand displacement activity to generate multiple copies, and repeating the nicking and extension rounds to amplify DNA.
Repeated strand-displacement nucleic-acid amplification around an inosine-substituted CpG
A method of amplifying a nucleic acid comprising binding a first primer containing an inosine base to one strand of DNA at a region having a CpG dinucleotide, extending the primer with an exonuclease deficient DNA polymerase to form a double stranded molecule having a first newly synthesized strand comprising an inosine base that substitutes for guanine in the CpG dinucleotide, nicking the first newly synthesized strand near the position of the inosine base, displacing the first newly synthesized strand with a DNA polymerase, and continuing the process to repeatedly form newly synthesized strands of DNA.
Multiple-copy template DNA amplification by nicking 3′ of incorporated inosine
A method for obtaining multiple copies of a template DNA strand comprising annealing a primer with an inosine base that substitutes for guanine in a CpG dinucleotide region, extending the 3′ end of the primer in the presence of an exonuclease deficient DNA polymerase to generate an extended primer comprising a primer portion and a first copy, generating a nick in the extended primer using an endonuclease that generates nicks 3′ of the inosine base, extending the primer region 5′ of the nick from the nick in the presence of the DNA polymerase to displace the 3′ portion and generate a second extended primer comprising a primer region and a second copy with an inosine base, and repeating the nicking and extending steps at least once to obtain multiple copies.
Incorporated inosine extension with endonuclease V nicking and repeating amplification steps
A method for amplifying a template DNA comprising annealing a primer containing an inosine base to the template DNA at a region having a CpG dinucleotide, extending the primer in the presence of a strand displacing DNA polymerase and deoxyinosine triphosphate to generate a first extension product comprising inosine with inosine substituting for guanine in the CpG dinucleotide, incubating the product with an endonuclease V to generate nicks in the first primer extension product at positions 3′ of the incorporated inosine, extending from the nicks with a strand displacing enzyme to generate second extension products, and repeating the endonuclease/nick-extension steps at least once to generate amplified template DNA.
The independent claims collectively protect an inosine-in-CpG, exonuclease deficient strand-displacement amplification approach in which amplification is driven by repeated primer extension, nick generation at or near the incorporated inosine, strand displacement of the inosine-containing primer, and successive rounds that produce amplified DNA substantially without thermal cycling.
Stated Advantages
Amplifies DNA substantially without thermal cycling.
Enables amplification using primers in connection with recognition and nicking of deoxyinosine in double-stranded DNA.
Potential to work directly on double-stranded DNA is stated in the document as described.
Potential use at field/ambient temperatures is stated in the document as described.
Documented Applications
Amplification of CpG dinucleotide-containing methylated targets, including specificity as described using CpG→TpG non-target.
Amplification of diluted human genomic DNA as described.
Amplification of HPV DNA as described.
Amplification support demonstrated for ribonucleotide-containing primers using RNase H as described.
Amplification support demonstrated with primers containing 8-deoxyguanine using Fpg and hOGG1 as described.
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