Method for the carry-over protection in DNA amplification systems targeting methylation analysis achieved by a modified pre-treatment of nucleic acids
Particular aspects provide methods for specific amplification of template DNA in the presence of potentially contaminating PCR products from previous amplification experiments. Particular embodiments comprise, in a first step, contacting DNA with a bisulfite solution, which sulfonates unmethylated (but not methylated) cytosines, resulting in cytosine deamination and generation of sulfonated uracil. Such sulfonation protects the template nucleic acid from being a target for the enzyme uracil-DNA-glycosylase (UNG), whereas any contaminating DNA, which contains unprotected unsulfonated or desulfonated uracils, is degraded enzymatically while the UNG is active. After UNG treatment and inactivation thereof, the sulfonated uracil bases are converted into uracil by desulfonation. Such aspects have substantial utility for decontamination of nucleic acid samples; e.g., for avoiding amplification of ‘carry over products’ in the context of DNA methylation analysis. In further aspects, the inventive methods can be generally used as simplified methods of bisulfite treatment.
1. A method for providing a decontaminated nucleic acid suitable for DNA 5-methylation analysis, comprising:
a) contacting a DNA sample with a bisulfite reagent under conditions suitable to produce a bisulfite-treated sulfonated DNA sample, wherein unmethylated cytosines of the DNA are sulfonated, sulfonated and deaminated, or comprise a mixture of both forms, and wherein said sulfonated, or sulfonated and deaminated uracil has not been significantly desulfonated; and
b) further contacting the bisulfite-treated sulfonated DNA sample with an amount of an enzyme that specifically degrades any contaminating nonsulfonated-uracil-containing nucleic acids and does not degrade sulfonated-uracil-containing nucleic acids thereby providing a decontaminated DNA sample suitable for DNA 5-methylation analysis.
2. The method of claim 1 , wherein the contaminating nonsulfonated-uracil-containing nucleic acids comprise nonsulfonated-uracil-containing carry-over contaminants from a prior nucleic acid amplification reaction, and wherein the decontaminated DNA sample is thereby optimized for use in a subsequent amplification analysis.
3. The method of claim 2 , wherein the subsequent amplification analysis comprises DNA methylation analysis.
4. The method of claim 1 , further comprising:
a) mixing the bisulfite-treated sulfonated DNA sample with at least one component required for a polymerase-mediated amplification reaction or an amplification-based detection assay;
b) inactivating, after specifically degrading any nonsulfonated-uracil-containing nucleic acids, the specifically degrading enzymatic activity; and
c) desulfonating, after said inactivating, the bisulfite-treated sulfonated DNA sample.
5. The method of claim 4 , wherein inactivating and desulfonating occur simultaneously by incubating the mixture at an increased temperature, said incubation sufficient to inactivate the specifically degrading enzymatic activity, and desulfonate the bisulfite-treated sulfonated DNA.
6. The method of claim 4 , further comprising, after desulfonating, amplifying the desulfonated DNA.
7. The method of claim 4 , wherein the specifically degrading enzyme is at least one selected from the group consisting of a DNA glycosylase and an endonuclease.
8. The method of claim 7 , wherein the DNA glycosylase is uracil-DNA-glycosylase (UNG).
9. The method of claim 4 , further comprising, after inactivating and desulfonating, performing a polymerase-based amplification reaction or an amplification-based assay.
10. The method of claim 9 , wherein the polymerase-based amplification reaction is initiated by a brief incubation at increased temperature, said incubation being sufficient to inactivate the specifically degrading enzymatic activity.
11. The method of claim 9 , wherein the polymerase is a heat stable polymerase.
12. The method of claim 9 , wherein the polymerase-mediated amplification, or the amplification-based assay is performed in the presence of dUTPs instead of dTTPs.
13. The method of claim 1 , wherein the amount of the specifically degrading enzyme is sufficient to degrade substantially all of any nonsulfonated-uracil-containing nucleic acid contaminants and does not degrade sulfonated-uracil-contaiing nucleic acids.
14. A method for providing decontaminated nucleic acid suitable for methylation analysis, comprising bisulfite treatment of a nucleic acid sample to provide for a sulfonated nucleic acid sample; and contacting the bisulfite-treated sulfonated nucleic acid sample with uracil-DNA-glycosylase (UNG) under conditions suitable to specifically degrade any contaminating nonsulfonated-uracil-containing nucleic acids to provide for a decontaminated nucleic acid sample suitable for methylation analysis thereby providing a decontaminated nucleic acid sample suitable for DNA 5-methylation analysis.