METHODS FOR ANALYSIS OF NUCLEIC ACID METHYLATION STATUS AND METHODS FOR FRAGMENTATION, LABELING AND IMMOBILIZATION OF NUCLEIC ACIDS

• US 20090233804A1
• Filed: 11/30/2007
• Published: 09/17/2009
• Est. Priority Date: 12/29/2003
• Status: Active Grant

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Dafforn, Geoffrey A., Kurn, Nurith
• Granted Patent: US 8,143,001 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites

Abstract

The invention relates to methods for analysis of nucleic acid methylation status, and fragmentation and/or labeling and/or immobilization of nucleic acids. More particularly, the invention relates to methods for fragmentation and/or labeling and/or immobilization of nucleic acids comprising labeling and/or cleavage and/or immobilization at abasic sites.

Claims

1-99. -99. (canceled)

100. A method for fragmenting a polynucleotide comprising a methylated nucleotide and an unmethylated nucleotide, said method comprising:

(a) cleaving a base portion of the unmethylated nucleotide from the polynucleotide with an agent capable of cleaving the base portion of an unmethylated nucleotide, whereby an abasic site is generated and wherein the agent is not capable of cleaving a base portion of a methylated nucleotide; and

(b) fragmenting the phosphodiester backbone of the polynucleotide at the abasic site, whereby polynucleotide fragments are generated.

101. The method of claim 100, wherein the agent capable of cleaving a base portion of an unmethylated nucleotide comprises an enzyme.

102. The method of claim 101, wherein the unmethylated nucleotide is cytosine and the enzyme comprises cytosine deaminase in conjunction with uracil DNA glycosylase.

103. The method of claim 100, wherein fragmentation of the phosphodiester backbone is performed with an agent selected from the group consisting of an enzyme, a chemical agent, acidic conditions, basic conditions, alkylating conditions, and heat.

104. The method of claim 103, wherein the agent for fragmenting the phosphodiester backbone comprises an amine.

105. The method of claim 104, wherein the amine is N,N′

-dimethylethylenediamine.

106. The method of claim 100, wherein the steps of the method are carried out simultaneously in the same reaction mixture.

107. The method of claim 100, wherein the polynucleotide is contacted with a methyl binding agent prior to, or during step (a).

108. The method of claim 107, wherein the methyl binding agent is an antibody.

109. The method of claim 107, wherein the methyl binding agent is a methyl binding protein.

110. The method of claim 100, wherein the polynucleotide is contacted with a polynucleotide binding protein prior to, or during step (a).

111. The method of claim 110, wherein the polynucleotide binding protein is a DNA binding protein.

112. The method of claim 111, wherein the DNA binding protein is a transcription factor or a regulatory protein.

113. The method of claim 100, wherein the polynucleotide has a native methylation status, and the fragments generated are related to the methylation status of the polynucleotide.

114. The method of claim 113, wherein the polynucleotide is hypermethylated, and the hypermethylated sequences are isolated.

115. The method of claim 100, further comprising:

(c) labeling at the abasic site, whereby a labeled polynucleotide fragment is generated.

116. The method of claim 115, wherein the labeled polynucleotide fragment comprises a detectable label.

117. The method of claim 116, further comprising hybridizing the labeled polynucleotide fragment to a probe.

118. The method of claim 117 wherein the probe is attached or hybridized to a solid support.

119. The method of claim 118, wherein the probe is attached to a microarray, attached to a bead array, or hybridized to a second probe attached to a solid support.

120. The method of claim 115, wherein the steps of the method are carried out simultaneously in the same reaction mixture.

121. The method of claim 115, wherein the polynucleotide is contacted with a methyl binding protein prior to or during step (a).

122. The method of claim 121, wherein the agent capable of cleaving the base portion of an unmethylated nucleotide is prevented from cleaving the base portion of a methylated nucleotide due to the binding of a methyl binding agent.

123. The method of claim 115 wherein the agent capable of cleaving a base portion of an unmethylated nucleotide comprises an enzyme.

124. The method of claim 123, wherein the unmethylated nucleotide is cytosine and the enzyme comprises cytosine deaminase in conjunction with uracil DNA glycosylase.

125. The method of claim 115, wherein the fragmenting of the phosphodiester backbone is performed by an amine.

126. A method for fragmenting a polynucleotide comprising a methylated nucleotide and an unmethylated nucleotide, the method comprising contacting the polynucleotide with a reaction mixture comprising:

(a) an agent capable of cleaving the base portion of an unmethylated nucleotide, wherein the agent is not capable of cleaving a base portion of a methylated nucleotide; and

(b) an agent capable of fragmenting the phosphodiester backbone of the polynucleotide.

127. The method of claim 126, the reaction mixture further comprising (c) an agent capable of labeling.

128. The method of claim 126, wherein the agent capable of cleaving a base portion of an unmethylated nucleotide comprises an enzyme.

129. The method of claim 128, wherein the unmethylated nucleotide is cytosine and the enzyme comprises cytosine deaminase in conjunction with uracil DNA glycosylase.

130. The method of claim 126, wherein the agent capable of fragmenting the phosphodiester backbone comprises an amine.

131. The method of claim 130, wherein the amine is N,N′

-dimethylethylenediamine.

132. A method for characterizing a polynucleotide that comprises a methylated nucleotide and an unmethylated nucleotide, the method comprising:

(a) contacting the polynucleotide with a reaction mixture comprising;

(i) an agent capable of cleaving the base portion of an unmethylated nucleotide, whereby an abasic site is generated and wherein the agent is not capable of cleaving a base portion of a methylated nucleotide;

(ii) an agent capable of fragmenting the phosphodiester backbone of the polynucleotide at the abasic site, whereby polynucleotide fragments are generated;

(iii) an agent capable of labeling at the abasic site, whereby labeled fragments are generated; and

(b) determining a methylation status of the polynucleotide by correlating methylation with the number, sizes, and/or composition of the labeled fragments generated in step (a).

133. The method of claim 132, wherein determining the methylation status comprises detecting the presence, absence, and/or quantity of methylation of the polynucleotide.

134. The method of claim 132, wherein the agent capable of fragmenting the phosphodiester backbone comprises an amine.

135. The method of claim 134, wherein the amine is N,N′

-dimethylethylenediamine.

136. The method of claim 132, wherein the agent cleaving a base portion of an unmethylated nucleotide comprises an enzyme.

137. The method of claim 136, wherein the unmethylated nucleotide is cytosine and the enzyme comprises cytosine deaminase in conjunction with uracil DNA glycosylase.

138. The method of claim 132, wherein a plurality of polynucleotides are characterized and compared.

139. The method of claim 138, wherein the plurality of polynucleotides compared represent different stages in development or different disease states.

140. The method of claim 138, wherein the each of the plurality of polynucleotides is labeled with a different label.

141. The method of claim 132, further comprising hybridizing the labeled polynucleotide fragments to a solid support or a probe.

142. The method of claim 141, wherein the labeled polynucleotide fragments are hybridized to a single array.

143. The method of claim 132, further comprising subtractive hybridization, wherein a first and a second polynucleotide are labeled with different labels, the first and second polynucleotides are mixed together, and the relative levels of methylation of the polynucleotides are determined.

144. A method of isolating a methylated portion of a polynucleotide that comprises a methylated nucleotide and an unmethylated nucleotide, the method comprising:

(a) treating the polynucleotide with a methyl binding agent;

(b) cleaving a base portion of an unmethylated nucleotide with an agent in a portion of the polynucleotide not bound to the methyl binding agent, whereby an abasic site is generated;

(c) fragmenting the phosphodiester backbone at the abasic site of the polynucleotide, whereby polynucleotide fragments are generated, and

(d) capturing the polynucleotide fragments generated in step (c).

145. The method of claim 144, wherein the agent capable of cleaving a base portion of an unmethylated nucleotide comprises an enzyme.

146. The method of claim 145, wherein said unmethylated nucleotide is cytosine and the enzyme comprises cytosine deaminase in conjunction with uracil DNA glycosylase.

147. The method of claim 144, wherein fragmenting the phosphodiester backbone is performed with an agent selected from the group consisting of an enzyme, a chemical agent, acidic conditions, basic conditions, alkylating conditions, and heat.

148. The method of claim 144, wherein fragmentation of the phosphodiester backbone is performed with an amine.

149. The method of claim 144, wherein fragmentation of the phosphodiester backbone is performed with an N,N′

-dimethylethylenediamine.

150. The method of claim 144, wherein the steps of the method are carried out simultaneously in the same reaction mixture.

151. The method of claim 144, wherein a polynucleotide captured in step (d) is captured on a microarray, a bead or by a probe in solution.