GENOME-WIDE qPCR
AssigneeThe Trustees of Columbia University in the City of New York
InventorsZhilun Zhao, Naixin Qian, Wei Min
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Abstract
A massively multiplexed qPCR technique that allows genome-wide qPCR (100-100,000 targets simultaneously) with few-hour turnaround and at lost cost. A massively multiplexed isothermal amplification technique that allows genome-wide multiplexed isothermal amplification (100-100,000 targets simultaneously) with few-hour turnaround and at lost cost. Microwell array chips, isothermal amplification imaging devices and qPCR imaging devices for use with the techniques are provided also.
Claims20
A microwell array (MWA) chip comprising a plurality of microwells,
- wherein each microwell contains a microbead that is covalently or non-covalently attached to at least one pair of primers comprising a forward primer and reverse primer, wherein the pair of primers is specific for a predefined nucleic acid, and wherein
- (a) each microbead comprises
- one or more types of Raman-active small molecule(s) (RASMs), each at a predefined concentration in the microbead, or
- one or more types of infrared-active (IR) small molecule(s) (IRASMs), each at a predefined concentration in the microbead;
- and
- (b) the Raman spectrum or IR spectrum of each microbead containing the one or more RASMs or IRASMs is individually resolvable from that of every other microbead in the plurality of microwells attached to a different at least one pair of primers.
A method of manufacturing a microwell array chip comprising
- printing a microwell array design on a soda lime photomask;
- spin-coating a photoresist layer onto a silicon wafer;
- exposing through the photomask, developing and hard-curing the photoresist layer on the silicon wafer so as to produce a microwell array-patterned silicon wafer;
- vacuum-depositing trichloro(1H, 1H,2H,2H-perfluorooctyl)silane onto the silicon wafer;
- pouring uncured and degassed PDMS polymer onto the patterned silicon wafer;
- and a) heat curing the PDMS polymer so as to form a PDMS microwell array chip;
- or b) placing a thin coverglass on top so that the PDMS polymer is sandwiched between the patterned silicon wafer and coverglass and applying pressure and heat to spread the PDMS across the coverglass substantially evenly and pushing the patterned silicon wafer to touch the coverglass, while heat for rapid curing so as to form a glass-bottomed PDMS microwell array chip;
- and
- removing the PDMS or glass-bottomed PDMS microwell array chip from the patterned silicon wafer;
- and
- adding to each microwell of the microwell array a predetermined microbead comprising one, or more, types of Raman-active small molecule(s) (RASMs), each at a predefined concentration in the microbead, or one or more, types of infrared-active (IR) small molecule(s) (IRASMs), each at a predefined concentration in the microbead, thereby producing a microwell array chip.
A method for determining the presence of and/or quantifying one or more predefined nucleic acids in a sample, the method comprising adding the sample to a microwell of a microwell array (MWA) chip comprising a plurality of microwells,
- wherein each microwell of the plurality has a microbead contained therein which has, covalently or non-covalently attached thereto, at least one pair of primers comprising a forward primer and a reverse primer specific for a predefined nucleic acid,
- and wherein the microbead of each microwell is individually resolvable from that of every other microbead in the plurality of microwells having a different at least one pair of primers,
- wherein the MWA chip contains a microwell for each predefined nucleic acid,
- and cleaving the covalent or non-covalent bond between each primer and the microbead and having therein provided, or adding, reagents sufficient to permit a polymerase chain reaction to occur therein, or reagents sufficient to permit an isothermal amplification reaction to occur therein, so as to produce one or more double-stranded amplicons of the predefined nucleic acid(s).
Dependencies are inferred from the claim text.