METHODS FOR FRAGMENTATION AND LABELING OF NUCLEIC ACIDS

• US 20100022403A1
• Filed: 07/02/2007
• Published: 01/28/2010
• Est. Priority Date: 06/30/2006
• Status: Abandoned Application

• Current Assignee: Nurith Kurn, Pengchin Chen
• Original Assignee: Nurith Kurn, Pengchin Chen
• Inventors: Chen, Pengchin, Kurn, Nurith
• Application Number:
  US12/305,633
• Publication Number:
  US 20100022403A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2533/101 Primer extension

Abstract

The invention provides methods, compositions, and kits for fragmentation and labeling of nucleic acids. More particularly, the invention relates to methods for fragmentation of nucleic acids to produce fragments with 3′ end hydroxyl groups within a desired size range. In methods of the invention, nucleic acids are fragmented at abasic sites to produce fragments with blocked 3′ ends. The 3′ ends are unblocked to produce polynucleotide fragments with hydroxyl groups at their 3′ ends. Methods, kits, and compositions for carrying out fragmentation of a polynucleotide template in a single reaction mixture to yield fragments with 3′-hydroxyl ends within the desired size range are disclosed.

Claims

1-74. -74. (canceled)

75. A method for generating a polynucleotide fragment, within a desired size range and comprising a hydroxyl group at the 3′

end, from a polynucleotide comprising an abasic site, said method comprising;

(a) contacting said polynucleotide comprising an abasic site with a reaction mixture comprising;

i. a chemical capable of fragmenting a phosphodiester backbone of a polynucleotide at an abasic site, whereby generating a polynucleotide fragment within the desired size range and comprising a blocked 3′

end; and

ii. an enzyme capable of unblocking the blocked 3′

end of said fragment, whereby generating a polynucleotide fragment within the desired size range and comprising a hydroxyl group at the 3′

end.

76. The method according to claim 75, wherein the chemical is a polyamine.

77. The method according to claim 76, wherein the polyamine is N,N′

-dimethylethylenediamine (DMED).

78. The method according to claim 75, wherein the enzyme capable of unblocking the blocked 3, end comprises a 3′

to 5′

exonuclease activity.

79. The method according to claim 78, wherein the enzyme comprises non-processive exonuclease activity and does not comprise an endonuclease activity.

80. The method according to claim 78, wherein the exonuclease activity is a non-processive exonuclease activity, wherein the enzyme that comprises an exonuclease activity also comprises an endonuclease activity, and wherein contacting the polynucleotide fragment with the enzyme is under conditions in which the endonuclease activity is minimized or absent.

81. The method according to claim 78, wherein the enzyme comprising a 3′

exonuclease activity is selected from the group consisting of endonuclease 4, exonuclease T, and apurinic/apyrimidinic endonuclease (APE

     1).

82. The method according to claim 75, further comprising:

(b) extending the polynucleotide fragment from the 3′

hydroxyl group with a polymerase.

83. The method according to claim 82, wherein the extension further comprises a labeled nucleotide, whereby a polynucleotide fragment within the desired size range and labeled at the 3′

end is generated.

84. The method of claim 83 wherein the polymerase is template independent.

85. The method according to claim 84, wherein the template independent polymerase is terminal deoxynucleotidyl transferase (TdT).

86. The method according to claim 83, wherein the labeled nucleotide is selected from the group consisting of a labeled nucleotide triphosphate (NTP), a labeled deoxynucleotide triphosphate (dNTP), and a labeled dideoxynucleotide triphosphate (ddNTP).

87. The method according to claim 83, wherein the labeled nucleotide is a biotinylated nucleotide.

88. The method according to claim 83, wherein the labeled nucleotide comprises a fluorophore.

89. The method according to claim 83, wherein a mixture of labeled and unlabeled nucleotides is used for labeling the polynucleotide fragment.

90. The method according to claim 75, wherein the reaction mixture further comprises:

iii. an agent capable of cleaving a base portion of a non-canonical nucleotide in a polynucleotide comprising a non-canonical nucleotide, whereby generating an abasic site;

or

iv. an agent capable of non-enzymatically converting a canonical or non-canonical nucleotide in a polynucleotide into an abasic site.

91. The method according to claim 90, wherein the non-canonical nucleotide is selected from the group consisting of dUTP, dITP, and 5-OH-Me-dCTP.

92. The method according to claim 90, wherein the agent capable of cleaving a base portion of the non-canonical nucleotide is an N-glycosylase enzyme.

93. The method according to claim 92, wherein the N-glycosylase is selected from the group consisting of Uracil N-Glycosylase (UNG), hypoxanthine-N-Glycosylase, and hydroxy-methyl cytosine-N-glycosylase.

94. The method according to claim 90, wherein the non-canonical nucleotide is dUTP and the enzyme capable of cleaving a base portion of the non-canonical nucleotide is UNG.

95. The method according to claim 90, wherein the non-canonical nucleotide is dUTP, the enzyme capable of cleaving a base portion of the non-canonical nucleotide is UNG, and the phosphodiester backbone is cleaved with DMED.

96. The method according to claim 90, wherein the polynucleotide comprising a non-canonical nucleotide is synthesized in the presence of two or more different non-canonical nucleotides, whereby a polynucleotide comprising two or more different non-canonical nucleotides is synthesized.

97. The method according to claim 90, wherein the polynucleotide comprising a non-canonical nucleotide is synthesized in the presence of all four canonical nucleotides and a non-canonical nucleotide, wherein the non-canonical nucleotide is provided at a ratio suitable for generating fragments within the desired size range.

98. The method according to claim 83 further comprising:

(c) characterizing a polynucleotide template of interest, comprising analyzing a polynucleotide fragment within the desired size range and labeled at the 3′

end.

99. The method according to claim 98, wherein analyzing the labeled polynucleotide fragment within the desired size range comprises determining amount of said products, whereby the amount of the polynucleotide template present in a sample is quantified.

100. The method according to claim 98, wherein analyzing the labeled polynucleotide fragment within the desired size range comprises contacting the labeled polynucleotide fragment with at least one probe.

101. The method according to claim 100, wherein the at least one probe is provided as a microarray.

102. The method according to claim 83 further comprising:

(c) determining a gene expression profile in a sample, said method comprising determining the amount of the polynucleotide fragment within the desired size range and labeled at the 3′

end wherein the amount is indicative of the amount of a polynucleotide template in said sample from which the labeled polynucleotide fragment was generated, whereby a gene expression profile is determined.

103. The method according to claim 102, wherein the polynucleotide template is RNA or mRNA.

104. The method according to claim 102, wherein the amounts of a plurality of polynucleotide fragments within the desired size range derived from a plurality of polynucleotide templates in a sample are determined.

105. The method according to claim 83 further comprising:

(c) hybridizing a first population of polynucleotide fragments within the desired size range and labeled at the 3′

end, to at least one probe.

106. The method according to claim 105 further comprising:

(d) comparing hybridization of the first population of polynucleotide fragments within the desired size range and labeled at the 3′

end to at least one probe with hybridization of a second population of polynucleotide fragments within the desired size range and labeled at the 3′

end to the at least one probe.

107. The method according to claim 83 further comprising:

(c) detecting presence or absence of a mutation in a template, comprising analyzing a polynucleotide fragment within the desired size range and labeled at the 3′

end, whereby presence of absence of a mutation is detected, wherein analyzing comprises comparison of the polynucleotide fragment within the desired size range and labeled at the 3′

end to a polynucleotide prepared from a reference polynucleotide.

108. The method according to claim 107, wherein the mutation is selected from the group consisting of a base substitution, a base insertion, a base deletion, and a single nucleotide polymorphism.

109. The method according to claim 75, wherein the polynucleotide comprising an abasic site is generated by cleaving a base portion of a methylated nucleotide with an agent capable of cleaving a base portion of the methylated nucleotide to create an abasic site, whereby an abasic site is generated.

110. The method according to claim 75, wherein the polynucleotide comprising an abasic site is generated by cleaving a base portion of a canonical nucleotide with an agent capable of cleaving a base portion of the canonical nucleotide to create an abasic site, whereby an abasic site is generated.

111. The method according to claim 110, wherein the canonical nucleotide is cytosine and the agent capable of cleaving a base portion of the canonical nucleotide comprises cytosine deaminase in conjunction with UNG.

112. The method according to claim 75, wherein the polynucleotide comprising an abasic site is synthesized from a polynucleotide template comprising DNA or RNA.

113. The method according to claim 112, wherein the polynucleotide template is selected from the group consisting of mRNA, cDNA, and genomic DNA.

114. The method according to claim 75, wherein the polynucleotide comprising an abasic site is single stranded or double stranded.

115. The method according to claim 75, wherein the polynucleotide comprising an abasic site is synthesized by an amplification method selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), multiple displacement amplification (MDA), rolling circle amplification (RCA), single primer isothermal amplification (SPIA), and Ribo-SPIA.

116. The method according to claim 75, wherein the polynucleotide comprising an abasic site is synthesized by a method selected from the group consisting of reverse transcription, primer extension, limited primer extension, replication, and nick translation.

117. A method for fragmenting a polynucleotide comprising an abasic site to generate fragments within a desired size range, said method comprising:

(a) chemically cleaving a phosphodiester backbone of a polynucleotide comprising an abasic site at the abasic site, whereby a polynucleotide fragment within the desired size range and comprising a blocked 3′

end is generated; and

(b) contacting the polynucleotide fragment with an enzyme capable of unblocking the blocked 3′

end of said fragment, whereby a polynucleotide fragment within the desired size range and comprising a 3′

end hydroxyl group is generated;

wherein (a) and (b) are performed simultaneously and performed in the same reaction mixture.

118. A composition or kit comprising:

(a) a chemical agent capable of cleaving a phosphodiester backbone at an abasic site to produce a polynucleotide fragment with a blocked 3′

end within a desired size range; and

(b) an enzyme capable of unblocking a blocked 3′

end to generate a polynucleotide comprising a 3′

hydroxyl group.

119. The composition or kit according to claim 118 wherein the composition or kit further comprises:

(c) an agent capable of cleaving a base portion of a nucleotide to generate an abasic site in a polynucleotide.

120. The composition or kit according to claim 118, wherein (a) is a polyamine, (b) is an enzyme comprising a 3′

exonuclease activity, and (c) is an N-glycosylase.

121. The composition or kit according to claim 120, wherein the polyamine is DMED, the enzyme comprising a 3′

exonuclease activity is selected from the group consisting of endonuclease 4, exonuclease T, and APE 1, and the N-glycosylase is UNG.

122. The kit according to claim 119, further comprising:

(d) an agent capable of labeling a 3′

hydroxyl group of a polynucleotide.

123. The composition or kit according to claim 119, further comprising:

(c) a non-canonical nucleotide; and

(d) an enzyme capable of synthesizing a polynucleotide comprising the non-canonical nucleotide.

124. The composition or kit according to claim 123, wherein the non-canonical nucleotide is dUTP and the agent capable of cleaving a base portion of a nucleotide to generate an abasic site in a polynucleotide is UNG.

125. The composition or kit according to claim 122, further comprising:

(e) a labeled nucleotide.

126. The composition or kit according to claim 125, wherein (a) is a polyamine, (b) is an enzyme comprising a 3, exonuclease activity;

(c) is an N-glycosylase (d) is a template independent polymerase; and

(e) is a biotinylated nucleotide.

127. The composition or kit according to claim 125, wherein the labeled nucleotide is selected from the group consisting of a labeled nucleotide triphosphate (NTP), a labeled deoxynucleotide triphosphate (dNTP), and a labeled dideoxynucleotide triphosphate (ddNTP).

128. The composition or kit according to claim 127, wherein the labeled nucleotide is a biotinylated nucleotide.

129. The composition or kit according to claim 125, wherein the polyamine is DMED, the enzyme comprising a 3′

exonuclease activity is selected from the group consisting of endonuclease 4, exonuclease T, and APE 1, the N-glycosylase is UNG, an agent capable of labeling a 3a hydroxyl group of a polynucleotide is TdT, and the labeled nucleotide is selected from the group consisting of biotin 2′

,3′

-dideoxy-UTP and biotin 2′

,3′

-dideoxy-CTP 17.

130. A population of single-stranded polynucleotide fragments of a desired size range representative of a complete genome or complete transcriptome of an organism wherein each of said fragments comprises a hydroxyl group at the 3′

end.

131. The population of claim 130 wherein the sizes of the fragments are about 50 to 200 nucleotides.