INTEGRATED PURIFICATION AND MEASUREMENT OF DNA METHYLATION AND CO MEASUREMENT OF MUTATIONS AND/OR MRNA EXPRESSION LEVELS IN AN AUTOMATED REACTION CARTRIDGE

摘要

In various embodiments 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 bisulfite 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) desulfonating the bound deaminated nucleic acid and/or simultaneously eluting and desulfonating the nucleic acid by contacting the deaminated nucleic acid with an alkaline solution to produce a bisulfite converted nucleic acid; vi) eluting said bisulfite 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.