Treatment of nucleic acid

Abstract

The invention provides methods for treating nucleic acid, particularly nucleic acid that is methylated. In one embodiment, the method can include the steps of (a) providing a denaturing environment to a nucleic acid sample; (b) reacting the nucleic acid sample with a bisulphite reagent and incubating the reaction so as to form a treated nucleic acid sample where methylated nucleotides in the nucleic acid sample remain unchanged while unmethylated nucleotides are converted to another form; (c) diluting the treated nucleic acid sample so as to reduce salt concentration to a level which will not substantially interfere with a nucleic acid precipitating step; (d) precipitating the diluted treated nucleic acid to substantially remove any unwanted reagents or diluents from treated nucleic acid; and (e) carrying out de-sulphonation of the precipitated treated nucleic acid so as to remove sulphonate groups present on the treated nucleic acid so as to obtain a nucleic acid sample substantially free of sulphonate groups.

Description

TECHNICAL FIELD [0001] The present invention relates to modified methods for treating nucleic acid, particularly methylated nucleic acid using bisulphite. BACKGROUND [0002] As a result of advances in automated sequencing technology, much work has been carried out on determining coding regions of DNA resulting in the full sequencing of many animal genomes including the human genome. It has been realised for many years that the majority of genomic DNA, however, is non-coding and this material was once considered as “junk” DNA. Analysis of the non-coding regions of DNA is now being considered as important in the study of gene expression and function. Methylation states or patterns in nucleic acid, particularly genomic DNA, is thought to have a functional or regulatory role in gene expression and control in animals. [0003] It has been demonstrated that, in single stranded DNA, sodium bisulphite preferentially deaminates cytosine to uracil, compared to a very slow rate of deamination of ...

AI Technical Summary

Benefits of technology

"The invention is a method for treating nucleic acids with a bisulphite reagent to modify methylated nucleotides with sulphonate groups. The method is efficient, adaptable for many different molecular biological techniques, and can achieve significant retention of nucleic acid. The method includes denaturing the nucleic acid sample, incubating it with the bisulphite reagent, diluting it, precipitating it, and reacting it to remove sulphonate groups. The method can be carried out without causing any substantial degradation or loss of the nucleic acid sample. The method can further be processed or analyzed. The kits containing reagents and instructions to carry out the method are also provided."

Problems solved by technology

In contrast, bisulphite methods presently in use or described in the prior art typically result in loss of up to about 96% of the nucleic acid sample so that only about 4% of the nucleic acid is actually available for analysis.

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