Integrated purification and measurement of DNA methylation and co-measurement of mutations and/or mRNA expression levels in an automated reaction cartridge

Abstract

Methods of determining methylation of DNA are provided. In one illustrative, but non-limiting embodiment the method comprises i) contacting a biological sample comprising a nucleic acid to a first matrix material comprising a first column or filter where said matrix material binds and / or filters nucleic acids in said sample and thereby purifies the DNA; ii) eluting the bound DNA from the first matrix material and denaturing the DNA to produce eluted denatured DNA; iii) heating the eluted DNA in the presence of bi sulfite ions to produce a deaminated nucleic acid; iv) contacting said deaminated nucleic acid to a second matrix material comprising a second column to bind said deaminated nucleic acid to said second matrix material; v) desulphonating the bound deaminated nucleic acid and / or simultaneously eluting and desulphonating the nucleic acid by contacting the deaminated nucleic acid with an alkaline solution to produce a bi sulfite converted nucleic acid; vi) eluting said bi sulfite converted nucleic acid from said second matrix material; and vii) performing methylation specific PCR and / or nucleic acid sequencing, and / or high resolution melting analysis (HRM) on said bisulfite-converted nucleic acid to determine the methylation of said nucleic acid, wherein at least steps iv) through vi) are performed in a single reaction cartridge.

Description

[0001] Cross References to Related Applications

[0002] This application claims the benefit of earlier US Provisional Application 62 / 433,165, filed December 12, 2016, which is hereby incorporated by reference in its entirety. Background technique

[0003] The genomes of higher eukaryotes contain modified 5-methylcytidine (5-meC). This modification usually occurs as part of a dinucleoside CpG in which cytosine is converted to 5-methylcytosine in a reaction that involves flipping a target cytosine out of the full double helix And the methyl group is transferred from S-adenosylmethionine by methyltransferases (see, eg, Klimasauskas et al. (1994) Cell 76:357-369). This enzymatic conversion is the major DNA epigenetic modification known to occur in vertebrates and is essential for normal embryonic development (see, e.g., Bird (1992) Cell 70:5-8; Laird and Jaenisch ( 1994) Human Mol. Genet. 3:1487-1495; and Li et al. (1992) Cell 69:915-926).

[0004] In eukaryotic cells, DNA met...

Images