Method and System for Multiplex Genetic Analysis

Abstract

The present disclosure provides apparatus, systems and method for detecting separately and substantially simultaneously light emissions from a plurality of localized light-emitting analytes. A system according to exemplary embodiments of the present disclosure comprises a sample holder having structures formed thereon for spatially separating and constraining a plurality of light-emitting analytes each having a single nucleic acid molecule or a single nucleic acid polymerizing enzyme, a light source configured to illuminate the sample holder, an optical assembly configured to collect and detect separately and substantially simultaneously light emissions associated with the plurality of light emitting analytes. The system may further include a computer system configured to analyze the light emissions to determine the structures or properties of a target nucleic acid molecule associated with each analyte.

1 - 45 . (canceled) 
     
     
         46 . A method for sequencing a plurality of target nucleic acid molecules, comprising:
 enriching a sample holder with a plurality of source points each having a single one of the target nucleic acid molecules and/or a single nucleic acid polymerizing enzyme molecule;   subjecting the plurality of source points to nucleic acid polymerization reactions, (1) wherein when the source points have a single one of the target nucleic acid molecules, the source points each further comprise fluorescence-labeled bases, primers, and at least one nucleic acid polymerizing enzyme molecule; (2) wherein when the source points have a single nucleic acid polymerizing enzyme molecule, the source points each further comprise fluorescence-labeled bases, primers, and at least one of the target nucleic acid molecules; and (3) wherein when the source points have a single one of the target nucleic acid molecules and a single nucleic acid polymerizing enzyme molecule, the source points each further comprise fluorescence-labeled bases and primers;   directing excitation light toward the sample holder at an angle with respect to a normal of the sample holder to illuminate the source points and to cause the source points to fluoresce; and   collecting fluorescent signals from the illuminated source points and focusing the fluorescent signals onto at least one detector to form images of the source points on the at least one detector to determine time sequences of base incorporations in the polymerization reactions.   
     
     
         47 . The method of  claim 46  wherein the at least one detector has multiple pixel elements and the optical assembly is configured to form spatially resolved images of the source points on the at least one detector. 
     
     
         48 . The method of  claim 47  wherein the sample holder comprises a plurality of housings and enriching the sample holder comprises:
 coating a bottom surface in each of the housings with a linker; 
 applying a dilute solution of polymerase molecules or a dilute solution of target nucleic acid molecules to the sample holder; 
 identifying a first group of housings each having at least one polymerase molecule or target nucleic acid molecule; 
 shining light onto each of the first group of housings to activate the linker and bind the at least one polymerase or target nucleic acid molecule to the bottom surface in the housing; 
 identifying an additional group of housings each having at least one polymerase molecule or target nucleic acid molecule; and 
 shining light onto each of the additional group of housings to activate the linker and bind the at least one polymerase or target nucleic acid molecule to the bottom surface in the housing. 
 
     
     
         49 . The method of  claim 48  further comprising controlling the concentration of the solution to be lower than an optimal Poisson distribution. 
     
     
         50 . The method of  claim 48  further comprising removing the solution and applying another dilute solution of polymerase molecules or another dilute solution of target nucleic acid molecules to the sample holder before identifying the additional group of housings. 
     
     
         51 . The method of  claim 48  further comprising repeating the steps of identifying an additional group of housings and shining light to the additional group of housings until a majority of the housings each has a single polymerase molecule or a single nucleic acid molecule bound to the bottom surface in the housing. 
     
     
         52 . The method of  claim 47  wherein the sample holder comprises a plurality of housings and enriching the sample holder comprises:
 providing a plurality of attachment sites on the sample holder; 
 applying a sample fluid comprising nanobeads to the sample holder, each nanobead being sized such that only one nanobead is likely to fit in a housing, at least a portion of the nanobeads in the sample fluid each having an enzyme or nucleic acid molecule attached thereto; 
 attaching an enzyme or nucleic acid molecule associated with a nanobead to each of a group of attachment sites on the sample holder; and 
 removing the nanobeads from the attached enzyme or nucleic acid molecules. 
 
     
     
         53 . The method of  claim 52  wherein each enzyme or nucleic acid molecules associated with a nanobead has a photoactivatable linker for linking with an attachment site and the step of attaching comprises:
 shining light onto the plurality of housings to activate the photoactivable linker. 
 
     
     
         54 . The method of  claim 52  wherein the enzyme or nucleic acid molecules are attached to the nanobeads by cleavable linkers and the removing the nanobeads comprises chemically cleaving the cleavable linkers. 
     
     
         55 . The method of  claim 52  wherein the removing the nanobeads comprises dissolving the nano beads. 
     
     
         56 . The method of  claim 47  wherein the sample holder has channels formed thereon and enriching the sample holder comprises:
 coating bottom surfaces of the channels with a photoactivatable linker; 
 applying a solution of polymerase molecules or target nucleic acid molecules to the sample holder; and 
 illuminating the bottom surfaces of the channels with a light pattern resulting in interleaving lighted and dark areas on the bottom surfaces of the channels to selectively activate the photoactivatable linker thereon. 
 
     
     
         57 . The method of  claim 56  wherein the light pattern is formed by interference or by using a grating. 
     
     
         58 . The method of  claim 47  wherein the sample holder has channels formed thereon and enriching the sample holder comprises:
 coating bottom surfaces of the channels with a photoactivatable linker; 
 applying a solution of oligonucleotide molecules to the sample holder; 
 stretching an oligonucleotide molecule along a bottom surface of each of a plurality of channels; and 
 illuminating the bottom surfaces of the plurality of channels with light to cause the oligonucleotide molecule in each of the plurality of channels to be held to the bottom surface of the channel. 
 
     
     
         59 . The method of  claim 58  wherein the stretching the oligonucleotide molecule comprises:
 attaching one end of the oligonucleotide molecule to an end of each of the plurality of channels; and 
 applying an electric field along each of the plurality of channels. 
 
     
     
         60 . The method of  claim 59  wherein applying the electric field comprises providing two electrodes of opposite polarity, one electrode being above the sample holder near one end of a channel and another electrode being near another end of the channel and below a bottom surface of the channel