Families of Non-Cross-Hybridizing Polynucleotides for Use as Tags and Tag Complements, Manufacture and Use Thereof

• US 20100311957A1
• Filed: 12/21/2009
• Published: 12/09/2010
• Est. Priority Date: 01/25/2001
• Status: Active Grant

• Current Assignee: Luminex Molecular Diagnostics Incorporated (DiaSorin S.p.A.)
• Original Assignee: Luminex Molecular Diagnostics Incorporated (DiaSorin S.p.A.)
• Inventors: Kobler, Daniel, Fieldhouse, Daniel
• Granted Patent: US 8,624,014 B2
• Time in Patent Office: Days
• US Class Current: 536/23.1
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C40B 20/04 Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

A family of minimally cross-hybridizing nucleotide sequences, methods of use, etc. A specific family of 1168 24mers is described.

Claims

1-95. -95. (canceled)

96. A composition comprising a set of minimally cross-hybridizing oligonucleotide tags or tag complements, each tag or tag complement in the composition either being free of cytosine residues and comprising guanosine residues or being free of guanosine residues and comprising cytosine residues,

wherein for each tag or tag complement that comprises cytosine residues, the cytosine residues are separated by between one and six non-cytosine residues, and for each tag or tag complement that comprises guanosine residues, the guanosine residues are separated by between one and six non-guanosine residues,

wherein the number of cytosine and guanosine residues present in each tag or tag complement in the composition does not exceed L/4 where L is the number of bases in the oligonucleotide,

wherein the length of each tag or tag complement in the composition differs by no more than five bases from the average length of all tags or tag complements in the composition,

wherein each tag or tag complement in the composition contains no more than 4 contiguous identical nucleotides,

wherein the number of guanosine and cytosine residues present in each tag or tag complement in the composition does not vary from the average number of guanosine and cytosine residues present in all other tags or tag complements of the composition by more than one, and

when each tag or tag complement of the composition is exposed to hybridization conditions comprising 0.2M NaCl, 0.1M Tris, 0.08% Triton X-100, pH 8.0 at 37°

C., the degree of cross-hybridization between the tag or tag complement and an oligonucleotide of the set that is not fully complementary to the tag or tag complement does not exceed 30% of the degree of hybridization between the tag or tag complement and a fully complementary oligonucleotide to said tag or tag complement.

97. The composition of claim 96, further characterized in that, each tag or tag complement in the composition contains either a cytosine or guanosine residue located within seven residues of an end of the tag or tag complement.

98. The composition of claim 96, wherein the length of each tag or tag complement in the composition is identical.

99. The composition of claim 98, wherein the number of guanosine and cytosine residues present in each tag or tag complement is the same.

100. The composition of claim 96, wherein the tags or tag complements are attached to a solid phase support.

101. The composition of claim 100, wherein the support is a planar substrate comprising a plurality of spatially addressable regions.

102. The composition of claim 96, wherein the tags or tag complements are covalently linked to microparticles.

103. The composition of claim 102, wherein the microparticles are spectrophotometrically unique and each unique microparticle has a different tag or tag complement attached thereto.

104. The composition of claim 96, wherein the degree of cross-hybridization between a said oligonucleotide and any complement of a different oligonucleotide of the composition does not exceed 10% of the degree of hybridization between said oligonucleotide and a complement to said oligonucleotide.

105. The composition of claim 96, wherein each oligonucleotide of the set is at least ten nucleotides in length.

106. A composition comprising minimally cross-hybridizing molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide comprising a sequence of ten to fifty nucleotide bases in length and for which, under a single set of hybridization conditions, the degree of cross-hybridization between a said oligonucleotide and any complement of a different oligonucleotide does not exceed about 20% of the degree of hybridization between said oligonucleotide and a complement to said oligonucleotide, and wherein each sequence is free of cytosine residues and comprises guanosine residues and, for each sequence, the number of guanosine residues does not exceed L/4 where L is the number of bases in said sequence.

107. The composition of claim 106, wherein each said sequence is of the same length as every other said sequence.

108. The composition of claim 107, wherein each said sequence is twenty-four bases in length.

109. The composition of claim 106, wherein the number of guanosine residues in each said sequence does not vary from the average number of guanosine residues in all of the sequences of the set by more than one.

110. The composition of claim 109, wherein each said sequence is twenty-four bases in length and each said sequence contains six guanosine residues.

111. The composition of claim 106, wherein at the 5′

-end of each said sequence at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence is a guanosine residue.

112. The composition of claim 106, wherein at the 3′

-end of each said sequence at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence is a guanosine residue.

113. The composition of claim 111, wherein at the 3′

-end of each said sequence at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence is a guanosine residue.

114. A composition of claim 106, wherein each said molecule is linked to a solid phase support so as to be distinguishable from a mixture of other said molecules by hybridization to its complement.

115. The composition of claim 114, wherein each molecule is linked to a location on a said solid phase support that is different than the location for each other different said molecule.

116. The composition of claim 115, wherein each said solid phase support is a microparticle and each said molecule is covalently linked to a different microparticle than each other different said molecule.

117. A composition comprising minimally cross-hybridizing molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide comprising a sequence of ten to fifty nucleotide bases in length and for which, under a single set of hybridization conditions, the degree of cross-hybridization between a said oligonucleotide and any complement of a different oligonucleotide does not exceed about 20% of the degree of hybridization between said oligonucleotide and a complement to said oligonucleotide, and wherein each sequence is free of guanosine residues and comprises cytosine residues and, for each sequence, the number of cytosine residues does not exceed L/4 where L is the number of bases in said sequence.

118. The composition of claim 117, wherein each said sequence is of the same length as every other said sequence.

119. The composition of claim 118, wherein each said sequence is twenty-four bases in length.

120. The composition of claim 117, wherein the number of cytosine residues in each said sequence does not vary from the average number of cytosine residues in all of the sequences of the set by more than one.

121. The composition of claim 120, wherein each said sequence is twenty-four bases in length and each said sequence contains six cytosine residues.

122. The composition of claim 117, wherein at the 5′

-end of each said sequence at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence is a cytosine residue.

123. The composition of claim 117, wherein at the 3′

-end of each said sequence at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence is a cytosine residue.

124. The composition of claim 122, wherein at the 3′

-end of each said sequence at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence is a cytosine residue.

125. A composition of claim 117, wherein each said molecule is linked to a solid phase support so as to be distinguishable from a mixture of other said molecules by hybridization to its complement.

126. The composition of claim 125, wherein each molecule is linked to a location on a said solid phase support that is different than the location for each other different said molecule.

127. The composition of claim 126, wherein each said solid phase support is a microparticle and each said molecule is covalently linked to a different microparticle than each other different said molecule.