US 9,175,340 B2Granted

Reactivity-dependent and interaction-dependent PCR

AssigneePresident and Fellows of Harvard College

InventorsDavid R. Liu, David Gorin, Adam Kamlet, Lynn M. McGregor, Christoph E. Dumelin

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Abstract

Methods, reagents, compositions, and kits for reactivity-dependent polymerase chain reaction (RD-PCR) and interaction-dependent polymerase chain reaction (ID-PCR) are provided herein. RD-PCR is a technique useful for determining whether a reactive moiety can form a covalent bond to a target reactive moiety, for example, in screening a library of candidate reactive moieties for reactivity with a target reactive moiety, and in identifying an enzyme substrate, for example, in protease substrate profiling. ID-PCR is a technique useful for determining whether a ligand can non-covalently bind to a target molecule, for example, in screening a library of candidate ligands for non-covalent interaction with a target molecule. RD-PCR and ID-PCR are also useful in detecting the presence of an analyte or an environmental condition.

Claims20

Dependent claims

A method for interaction-dependent polymerase chain reaction, comprising:

(i) providing a nucleic acid template, wherein the nucleic acid template comprises:
a first primer hybridization site,
optionally, a sequence tag,
a second primer hybridization site, and
a candidate ligand;
(ii) contacting the nucleic acid template with a first primer, wherein the first primer comprises:
a sequence complementary to the first primer hybridization site,
a third primer hybridization site, and
a target molecule;
(iii) incubating the nucleic acid template contacted with the first primer under conditions suitable for the candidate ligand to bind to the target molecule with a KD<10−6;
(iv) incubating the nucleic acid template contacted with the first primer under conditions suitable for bound first primer to hybridize with the first primer hybridization site of the nucleic acid template it is bound to and for primer extension, and not allowing for efficient primer site hybridization and primer extension of the first primer not connected to the nucleic acid template by an interaction characterized by a KD<10−6;
(v) contacting the nucleic acid template contacted with the first primer with a PCR primer complementary to the second primer hybridization site and a PCR primer complementary to the third primer hybridization site or with a PCR primer complementary to the second and third primer hybridization site;
and
(vi) performing a polymerase chain reaction to amplify the nucleic acid template or a fragment thereof.

11 more elements

A method for interaction-dependent polymerase chain reaction, comprising:

(i) providing a nucleic acid template, wherein the nucleic acid template comprises
a first primer hybridization site,
optionally, a sequence tag,
a second primer hybridization site, and
a ligand;
(ii) contacting the nucleic acid template with a first primer, wherein the first primer comprises:
a sequence complementary to the first primer hybridization site,
a third primer hybridization site, and
a binding molecule;
(iii) incubating the nucleic acid template contacted with the first primer under conditions suitable for the ligand to non-covalently bind to the binding molecule with a KD<10−6 either only in the presence or only in the absence of an environmental factor or analyte;
(iv) incubating the nucleic acid template contacted with the first primer under conditions suitable for bound first primer to hybridize with the first primer hybridization site of the nucleic acid template it is bound to and for primer extension, and not allowing for efficient primer site hybridization and primer extension of the first primer not connected to the nucleic acid template by an interaction characterized by a KD<10−6;
(v) contacting the nucleic acid template contacted with the first primer with a PCR primer complementary to the second primer hybridization site and a PCR primer complementary to the third primer hybridization site or with a PCR primer complementary to the second and third primer hybridization site;
and
(vi) performing a polymerase chain reaction to amplify the nucleic acid sequence, or a fragment thereof.

11 more elements

Dependencies are inferred from the claim text.

Facts

Application: 13/505,872Filed 13 Oct 2010 · us provisional application
Priority: 4 Nov 2009
Published: US 2013/0225426 A129 Aug 2013
Granted: 3 Nov 2015
Term ends: Not estimatedThe source records don’t support an estimate.

Classification5 codes

C12Q 1/686
Nucleic acid amplification reactions: polymerase chain reaction [PCR]
C12N 15/1075
Isolating an individual clone by screening libraries: by coupling phenotype to genotype, not provided for in other groups of this subclass
C12Q 1/6853
Nucleic acid amplification reactions: using modified primers or templates
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms