METHODS AND COMPOSITIONS FOR HIGH-THROUGHPUT BISULPHITE DNA-SEQUENCING AND UTILITIES
First Claim
1. A method for determining the epigenomic status of a target DNA population comprising:
- fragmenting the target DNA population into a suitable size to which one or more different modified-adaptors of a composition comprising a modified nucleotide substituting for its unmodified nucleotide analog at one or more or all positions, are ligated to the target DNA to yield a composition comprising;
(1) an identical modified-adaptor ligated to both ends of the target DNA fragment;
or(2) a different modified-adaptor ligated to each end of the target DNA fragment;
subjecting the modified-adaptor-ligated target DNA to a chemical treatment in which the target DNA composition and the modified-adaptor composition are rendered chemically and functionally distinguishable wherein the ligated modified adaptor or adaptors are essentially and functionally unaltered;
amplifying the chemically treated modified-adaptor-ligated DNA at least one round using primers that are complementary to sequences of the modified adaptor; and
sequencing the amplified DNA.
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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™, SOLiD™ or 454™-type DNA sequencing platforms to study DNA methylation.
25 Citations
21 Claims
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1. A method for determining the epigenomic status of a target DNA population comprising:
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fragmenting the target DNA population into a suitable size to which one or more different modified-adaptors of a composition comprising a modified nucleotide substituting for its unmodified nucleotide analog at one or more or all positions, are ligated to the target DNA to yield a composition comprising; (1) an identical modified-adaptor ligated to both ends of the target DNA fragment;
or(2) a different modified-adaptor ligated to each end of the target DNA fragment; subjecting the modified-adaptor-ligated target DNA to a chemical treatment in which the target DNA composition and the modified-adaptor composition are rendered chemically and functionally distinguishable wherein the ligated modified adaptor or adaptors are essentially and functionally unaltered; amplifying the chemically treated modified-adaptor-ligated DNA at least one round using primers that are complementary to sequences of the modified adaptor; and sequencing the amplified DNA. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method for the production of a hemi-methylated DNA control template to monitor bisulphite reaction efficiency comprising:
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providing complementary DNA strands, strand-A and strand-B, wherein the deoxycytosines of strand-A are methylated at the 5-carbon position, and wherein the deoxycytosines of strand-B are not methylated; and annealing a DNA strand-A to a DNA strand-B; strand-A having been created in a first amplification reaction comprising primer-A and primer-B, whereby primer-A deoxycytosines are substituted with 5-methyl-deoxycytosines and primer-B is labeled with biotin and DNA amplification is performed in the presence of a deoxyribonucleotide triphosphate mixture comprising of dATP, dTTP, dGTP, and 5-methyl-dCTP; and strand-B having been created in a second amplification reaction using the same template as the first reaction and comprising primer-A and primer-B, whereby primer-A is labeled with biotin and DNA amplification is performed in the presence of a deoxyribonucleotide triphosphate mixture comprising of dATP, dTTP, dGTP and dCTP; and
whereinequal molar amounts of double strand products of the first and the second amplification reactions are combined, denatured, allowed to re-anneal and are then subjected to avidin affinity chromatography to remove any undesired products.
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21. A method for the production of a methylated DNA control template wherein the deoxycytosines of both strands are methylated at the 5-carbon position to monitor bisulphite reaction efficiency comprising:
providing a control DNA template; and
amplifying that DNA in the presence of a deoxyribonucleotide triphosphate mixture comprising dATP, dTTP, dGTP, and 5-methyl-dCTP using primers where constituent deoxycytosines are substituted with 5-methyl-deoxycytosines.
Specification