Methods And Compositions For High-Throughput Bisulphite Dna-Sequencing And Utilities

Abstract

The Invention Relates To Novel Methods And Compositions To Produce Dna Templates Suitable For Chemical Modifications And High-Throughput Dna-Sequencing. A Method Of The Invention Relates To A Dna Adaptor Design Where Constituent Deoxycytosines Are Substituted With 5-Methyl-Deoxycytosines Rendering The Resulting Adaptor Resistant To Bisulphite Mediated Deamination. When Said Adaptor Is Ligated Onto Double Stranded Dna Template, Subsequent Dna Denaturation And Bisulphite Treatment Deaminates Template Dna Deoxycytosine Differentially To Deoxyuraeil Whilst The 5-Methyl-Deoxycytosines Of The Ligated Adaptor Resist Chemical Conversion Resulting In The Adaptor Sequence Remaining Unaltered. Both Strands Of Bisulphite Treated Dna Can Thus Be Amplified With A Single Primer Set That Hybridizes To The Unaltered Adaptor Sequence. The Invention Also Relates To Methods To Produce Control Template Of A Defined Methylation Composition To Optimize Conditions For The Bisulphite Reaction. In A Preferred Embodiment, The Present Invention Can Be Used To Produce Templates Suitable For Genome-Wide Bisulphite-Dna Sequencing Using Conventional, Solexa(Tm), Solid(Tm) Or 454(Tm)-Type Dna Sequencing Platforms To Study Dna Methylation.