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 bisulfite treatment of a nucleic acid, comprising:
a. contacting a nucleic acid with a bisulfite reagent under conditions suitable to produce a bisulfite-treated sulfonated nucleic acid sample, wherein unmethylated cytosines of the nucleic acid 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;
b. contacting the bisulfite-treated sulfonated nucleic acid sample with at least one component required for a polymerase-mediated amplification reaction or an amplification-based detection assay before desulfonating the sample, wherein the component is a heat stable polymerase; and
c. desulfonating the bisulfite-treated sulfonated nucleic acid sample in the presence of the polymerase by a brief incubation at an increased temperature.
2. The method of claim 1 , further comprising, after desulfonating, amplifying the desulfonated nucleic acid with a polymerase-mediated amplification reaction or an amplification-based detection assay.
3. The method of claim 2 , wherein the polymerase-based amplification reaction is briefly incubated at an increased temperature to simultaneously start the reaction, and allow for desulfonation of the bisulfite-treated sulfonated nucleic acid sample.