Enzymes for amplification and copying bisulphite modified nucleic acids
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Publication Number
US-8685675-B2
Publication Date
2014-04-01
Expiration Date
Abstract
The invention relates to the use of enzymes for copying or amplifying bisulphite modified or treated nucleic acids, wherein the enzymes are more effective in copying or amplifying the nucleic acid compared with native Taq polymerase under substantially the same conditions.
Core Innovation
The invention provides enzymes and methods for copying or amplifying bisulphite treated nucleic acid more effectively than native Taq polymerase while using less or no errors. The method includes bisulphite treating a nucleic acid and then copying or amplifying the bisulphite treated nucleic acid using an enzyme Pol A DNA polymerase variant 5D4 (5D4).
The problem addressed is bisulphite-induced damage that limits copying or amplification, including abasic sites and bulky sulphonate/adduct lesions formed during bisulphite treatment, which reduce copying and amplification efficiency and can limit the ability to support longer amplicons. The invention is directed to bypassing these lesions by using 5D4 rather than Taq polymerase under similar conditions to achieve improved fidelity and amplification efficiency.
In addition to 5D4, the described copying/amplifying enzymes include HIV-RT and variants, and other polymerase, reverse transcriptase, and endonuclease candidates, where examples are described that can copy bisulphite-treated RNA and improve extension and copying over damaged bisulphonated templates. The bisulphite workflow emphasizes conversion behavior where methylated cytosines remain unchanged while unmethylated cytosines are converted to uracil, with an optional desulphonation step described in the document.
Claims Coverage
The independent claim covers a method for copying or amplifying bisulphite treated nucleic acid using enzyme Pol A DNA polymerase variant 5D4 (5D4) with improved copying and amplification effectiveness and reduced errors compared to Taq polymerase. Based on the provided partial claim set, only one independent claim is explicitly identified.
Copying or amplifying bisulphite treated nucleic acid with 5D4
A method for copying or amplifying bisulphite treated nucleic acid comprising bisulphite treating a nucleic acid, and copying or amplifying the bisulphite treated nucleic acid using enzyme Pol A DNA polymerase variant 5D4 (5D4) wherein 5D4 copies or amplifies bisulfite modified nucleic acid more effectively with less or no errors compared to Taq polymerase.
The claim coverage centers on using Pol A DNA polymerase variant 5D4 (5D4) to copy or amplify bisulphite treated nucleic acid, framed as improved effectiveness and reduced errors relative to Taq polymerase. Dependent claims refine the nucleic acid and damage context, bisulphite chemistry, omission of desulphonation, conversion behavior, and amplification modalities including PCR, RT-PCR, qPCR, isothermal amplification, and signal amplification.
Stated Advantages
Copies or amplifies bisulfite modified nucleic acid more effectively than Taq polymerase.
Uses less or no errors compared to Taq polymerase.
Improves ability to extend and copy sulphonated templates with better fidelity and amplification efficiency versus Taq, including longer PCR product performance.
Documented Applications
Determining sequence, methylation, source, and detecting microorganisms using bisulphite workflow and downstream processing.
Amplifying bisulphite treated nucleic acid using PCR, RT-PCR, qPCR, isothermal amplification, or signal amplification formats.
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