Nucleic acid detection using degradation of a tagged sequence

• US 6,514,700 B1
• Filed: 06/21/2000
• Issued: 02/04/2003
• Est. Priority Date: 04/30/1999
• Status: Expired due to Fees

• Current Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Aclara Biosciences, Inc (Laboratory Corporation Of America Holdings)
• Inventors: Singh, Sharat
• Primary Examiner(s): Benzion, Gary
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/602,586
• Time in Patent Office: 958 Days
• Field of Search: 435/6, 435/91.2, 536/22.1
• US Class Current: 435/6.12
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C40B 20/08 Direct analysis of the library members per se by physical methods, e.g. spectroscopy
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/16 involving encoding steps
• CPC Class Codes: C40B 70/00 Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes

Abstract

Methods and compositions are provided for detecting target molecules, e.g. DNA sequences, particularly single nucleotide polymorphisms, using a pair of nucleotide sequences, a primer and a snp detection sequence, where the snp detection sequence binds downstream from the primer to the target DNA in the direction of primer extension, or ligands and receptors. The methods employ e-tags comprising a mobility-identifying region joined to a detectable label and a target-binding region. The result of the binding of the target-binding region to the target is to have a bond cleaved in the starting material with the production of a detectable product with a different mobility from the starting material, where the different e-tags can be separated and detected.

Claims

1. A method for detecting at least one target nucleic acid sequence in a nucleic acid sample, said method comprising:

combining said nucleic acid sample and at least one reagent pair consisting of a primer and an e-tag linked by a cleavable bond to a target-binding sequence, where at least one nucleotide linkage at positions immediately 3′

of the second nucleotide at the 5′

-end of said target-binding sequence is resistant to nuclease hydrolysis, each reagent pair having a sequence homologous to each nucleic acid sequence to be determined, wherein each said primer specifically binds to said target nucleic acid and said target-binding sequence binds to said target nucleic acid downstream from said primer, wherein each said target-binding sequence is characterized by being linked to a non-oligomeric e-tag specific for each said nucleic acid sequence;

executing at least one cycle of cleavage of said cleavable bond, whereby said e-tag is released substantially free of said target-binding sequence, such that cleavage of the target-binding sequence at its 5′

end produces a single released product comprised of the e tag and the 5′

-end nucleotide of the target-binding sequence, wherein each said released product produced from a given target-binding sequence has a known, unique electrophoretic mobility with respect to the released products produced from all other such target-binding sequences, by virtue of a unique charge/mass ratio associated with the e tag;

separating released products into individual fractions of released products specific for each target nucleic acid sequence; and

detecting said released product fractions,

whereby the presence in said target nucleic acid sample of said at least one nucleic acid sequence is detected.

2. A method according to claim 1, wherein said combining further comprises a 5′

-3′

-exonuclease to achieve said cleavage.

3. A method according to claim 1, wherein said primer further comprises a cleavage reagent on said primer to achieve said cleavage.

4. A method according to claim 3, wherein said cleavage reagent is an enzyme that produces singlet oxygen or hydrogen peroxide and said cleavable bond is oxidatively cleaved.

5. A method according to claim 1 wherein at least one link in said nucleic acid sequence of said target-binding sequence is resistant to nuclease cleavage.

6. A method according to claim 1, wherein a ligand is bonded to said e-tag linked target binding sequence where upon release of said e-tag, said ligand is retained with the remaining portion of said target-binding sequence, and further including the step after said combining, binding said ligand with a receptor to diminish any interference in said separating from ligand containing components.

7. A method according to claim 1, wherein said at least one target nucleic acid sequence comprises a snp and said cleavage is achieved by a 5′

-3′

exonuclease, wherein either said target-binding sequence or said primer includes a nucleotide complementary to said snp.