Method for accurate assessment of dna quality after bisulfite treatment
AssigneeSequenom, Inc.
InventorMathias EHRICH
Life of this patent
Estimating the term…
Abstract
The present invention is directed to methods useful for determining DNA quality after bisulfite treatment. The methods include a PCR-based assay, which allows ab-initio assessment of the DNA quality after bisulfite treatment and can help to prevent inaccurate quantitative measurement resulting from poor bisulfite treatment.
Claims19
A method to determine the maximum amplicon size for nucleic acid in a sample after bisulfite treatment that will yield accurate quantitative measurements, comprising:
- a) adding to a sample control nucleic acid having a known methylation ratio, thereby generating a sample comprising i) sample nucleic acid and ii) control nucleic acid;
- b) treating the sample with bisulfite;
- c) performing PCR using a primer set that generates at least two amplicons from a control region in the sample nucleic acid, wherein the amplicons vary in length and share a subset of CpG sites;
- d) determining amplification success and statistical variability for at least three CpG sites that are common to all of the amplicons of step (c);
- and
- e) selecting amplicon sizes that yield detectable amplification products and low statistical variability for a given sample.
A method to determine methylation conditions which yield results more accurate across a range of amplicon sizes for nucleic acid in a sample, comprising:
- a) adding to a sample control nucleic acid having a known methylation ratio, thereby generating a sample comprising i) sample nucleic acid and ii) control nucleic acid;
- b) treating the sample with bisulfite;
- c) performing PCR using a primer set that generates at least two amplicons from a control region in the sample nucleic acid, wherein the amplicons vary in length and share a subset of CpG sites;
- d) modifying at least one of the methylation conditions to introduce variable methylation conditions, wherein the methylation conditions are selected from the group consisting of sample handling, bisulfite treatment methods, amplification conditions, and methylation detection methods;
- e) determining amplification success and statistical variability for at least three CpG sites that are common to all of the amplicons of step (c);
- and
- f) selecting methylation conditions that are optimal across a range of amplicon sizes and yield detectable amplification products and low statistical variability for a given sample.
Dependencies are inferred from the claim text.