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...

AI Technical Summary

Problems solved by technology

There are currently no methylation kits that allow the user to complete the entire process in one step—DNA purification, bisulfite incubation, desulfonation, second DNA purification, and methylation-specific PCR

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