Devices, processes, and systems for determination of nucleic acid sequence, expression, copy number, or methylation changes using combined nuclease, ligase, polymerase, and sequencing reactions

Abstract

The present invention relates to methods, devices, instruments, processes, and systems for the highly specific, targeted molecular analysis of regions of human genomes and transcriptomes from the blood, i.e. from cell free circulating DNA, exosomes, microRNA, lncRNA, circulating tumor cells, or total blood cells. The technology enables highly sensitive identification and enumeration of mutation, expression, copy number, translocation, alternative splicing, and methylation changes using spatial multiplexing and combined nuclease, ligation, polymerase, and sequencing reactions. Such technology may be used for non-invasive early detection of cancer, non-invasive cancer prognosis, and monitoring both treatment efficacy and disease recurrence of cancer.

1 - 54 . (canceled) 
     
     
         55 . A method for preparing a system for identifying a plurality of nucleic acid molecules in a sample, said method comprising:
 providing the system comprising:
 (i) an inlet port and 
 (ii) a cartridge defining a space containing:
 multiple primary reaction chambers fluidically coupled to said inlet port to receive material from said inlet port and produce primary reaction chamber products from the material; 
 a product capture housing enclosing a solid support with a plurality of separate columns of a plurality of product capture subdivisions configured in separate rows and columns, with each separate product capture subdivision comprising an array of a plurality of individual hydrophilic micro-pores or micro-wells, wherein each of said individual hydrophilic micro-pores or micro-wells is separated by hydrophobic surfaces where primary reaction products are further reacted to create array products which are detected in the micro-pores or micro-wells, wherein one or more of the columns of separate product capture subdivisions receive material which has passed through one of said multiple primary reaction chambers; 
 multiple secondary reaction chambers, one or more of which are fluidically coupled to one of said multiple primary reaction chambers to receive material from one of said multiple primary reaction chambers; and 
 multiple mixing chambers each fluidically coupled to one of said multiple secondary reaction chambers to receive material from one of said multiple secondary reaction chambers and to discharge material to said product capture housing so that each column of separate product capture subdivisions is fluidically coupled to one of said multiple mixing chambers to receive material from one of said mixing chambers and 
 
   applying universal tag or capture oligonucleotide primers or probes to the micro-pores or micro-wells of the product capture subdivisions on the solid support within said product capture housing, whereby the universal tag or capture oligonucleotide primers or probes are retained within the micro-pores or micro-wells.   
     
     
         56 . The method of  claim 55  further comprising:
 filling said multiple primary reaction chambers and/or secondary reaction chambers with primary or secondary reaction oligonucleotide probes or primers each having a first portion comprising a nucleotide sequence complementary to a portion of target nucleic acids in the sample. 
 
     
     
         57 . The method of  claim 56 , wherein the primary or secondary reaction oligonucleotide probes or primers further comprise a second portion comprising a nucleotide sequence which is the same as or complementary to a portion of a universal tag or capture oligonucleotide primers, retained within the micro-pores or micro-wells. 
     
     
         58 . The method of  claim 55 , wherein each of said product capture subdivisions comprises an array of individual micro-pores each having opposed first and second open ends with the first end having a large diameter and the second end having a diameter which is smaller than that of the first end with a first passage in fluid communication with first end of the micro-pores and a second passage in fluid communication with the second end of the micro-pores, wherein the universal tag or capture oligonucleotide primers or probes are applied to the micro-pores by a method comprising the following steps in the sequence set forth as follows:
 passing the universal tag or capture oligonucleotide primers or probes through the first passage into the micro-pores through their first open ends while hydrophobic liquid is passed through the second passage;   passing a hydrophobic liquid through the first passage while the hydrophobic liquid is passed through the second passage;   passing a volatile solvent through the first passage while the hydrophobic liquid is passed through the second passage; and   passing air through the first passage while heat, a hydrophobic liquid, a volatile solvent, and then air is passed through the second passage.   
     
     
         59 . The method of  claim 55 , wherein each of said product capture subdivisions comprises an array of individual micro-pores each having opposed first and second open ends with the first end having a large diameter and the second end having a diameter which is smaller than that of the first end with a first passage in fluid communication with first end of the micro-pores and a second passage separated from the second end of the micro-pores by a mesh screen covering the second ends, in fluid communication with a second passage, wherein the detection or capture oligonucleotide primers or probes are applied to the micro-pores by a method comprising the following steps in the sequence set forth as follows:
 passing the universal tag or capture oligonucleotide primers or probes through the first passage into the micro-pores through their first open ends;   passing a hydrophobic liquid through the first passage to expel the universal tag or capture oligonucleotide primers or probes from the first passage;   passing a hydrophobic liquid through the first passage while a hydrophobic liquid is passed through the second passage;   passing a volatile solvent through the first passage while a hydrophobic liquid is passed through the second passage; and   passing air through the first passage while heat, a hydrophobic liquid, a volatile solvent, and then air is passed through the second passage.   
     
     
         60 . A process of identifying a plurality of nucleic acid molecules in a sample using a system comprising:
 (i) an inlet port and   (ii) a cartridge defining a space containing:
 multiple primary reaction chambers fluidically coupled to said inlet port to receive material from said inlet port and produce primary reaction chamber products from the material and 
 a product capture housing enclosing a solid support with a plurality of separate columns of a plurality of product capture subdivisions configured in separate rows and columns, with each separate product capture subdivision comprising an array of a plurality of individual hydrophilic micro-pores or micro-wells, wherein each of said individual hydrophilic micro-pores or micro-wells is separated by hydrophobic surfaces where primary reaction products are further reacted to create array products which are detected in the micro-pores or micro-wells, wherein one or more of the columns of separate product capture subdivisions receive material which has passed through one of said multiple primary reaction chambers; 
   applying universal tag or capture oligonucleotide primers or probes to the micro-pores or micro-wells of the product capture subdivisions on the solid support within said product capture housing, whereby the universal tag or capture oligonucleotide primers or probes are retained within the micro-pores or micro-wells;   filling said multiple primary reaction chambers with primary reaction chambers with primary reaction oligonucleotide probes or primers each having a first portion comprising a nucleotide sequence complementary to a portion of target nucleic acids in the sample;   conducting the primary reaction in said system; and   obtaining the nucleotide sequence of target nucleic acid molecules in the sample following said conducting the primary reaction.   
     
     
         61 . The process of  claim 60 , wherein said conducting the primary reaction comprises:
 providing a sample comprising a plurality of target nucleic acid molecules;   contacting the sample with a set of primary oligonucleotide primers having a first portion complementary to a portion of the target nucleic acid molecules and a second portion and a polymerase to form a polymerase chain reaction mixture;   subjecting said polymerase chain reaction mixture to a polymerase chain reaction in the multiple primary reaction chambers to produce a set of amplification products; and   passing the amplification products to said product capture housing enclosing a solid support with a plurality of separate columns of a plurality of capture subdivisions with each separate product capture subdivision comprising an array of a plurality of individual micro-pores containing immobilized captures probes complementary to the second portion, wherein said obtaining the nucleotide sequence of target nucleic acid molecules in the sample is carried out in said micro-pores.   
     
     
         62 . The process of  claim 61 , wherein the product capture subdivisions comprise an array of a plurality of individual micro-pores each having opposed first and second open ends with the first end having a large diameter and the second end having a diameter which is smaller than that of the first end. 
     
     
         63 . The process of  claim 62  further comprising:
 a mesh screen covering the second ends of the micro-pores in said product capture housing. 
 
     
     
         64 . The process of  claim 61  further comprising:
 a bead containing said immobilized capture probes placed in the individual micro-pores. 
 
     
     
         65 . The process of  claim 61  further comprising:
 removing at least one second portion from said amplification product before said obtaining the nucleotide sequence and after said subjecting said polymerase chain reaction mixture to a polymerase chain reaction. 
 
     
     
         66 . The process of  claim 65 , wherein said removing is carried out with uracil DNA glycosylases, apurinic/apyrimidinic endonuclease, endonuclease III, endonuclease IV, endonuclease V, alkyladenine DNA glycosylase, formamidopyrimidine DNA glycosylase, or 8-oxyguanine DNA glycosylase, or combinations thereof. 
     
     
         67 . The process of  claim 60 , wherein each of said product capture subdivisions comprises an array of individual micro-pores or micro-wells each having opposed first and second open ends with the first end having a large diameter and the second end having a diameter which is smaller than that of the first end with a first passage in fluid communication with first end of the micro-pores and a second passage in fluid communication with, and separated from, the second end of the micro-pores by a mesh screen covering the second ends of the micro-pores and wherein said obtaining the nucleotide sequence is carried by a process comprising the following steps in the sequence set forth as follows:
 passing the products of a polymerase chain reaction into said product capture housing through said first passage;   passing hydrophobic liquid through said first passage, such that the products are distributed into individual micro-wells;   passing hydrophobic liquid through said first and second passages;   amplifying said products in a polymerase chain reaction and/or isothermal reaction using the capture oligonucleotide primers under conditions to generate amplification products that are immobilized to the interior surface of the micro-wells;   passing a volatile solvent through said first passage while passing hydrophobic liquid through said second passage;   denaturing the products of the polymerase chain reaction and/or isothermal reaction and washing away non-anchored nucleic acid molecules through the first passage while passing hydrophobic liquid through said second passages, such that the products are isolated in individual micro-wells;   passing hydrophobic liquid with a higher density than water through said first passages while passing volatile solvent, air, and then sequencing reagents through the second passages; and   carrying out a sequencing reaction in said product capture subdivision.   
     
     
         68 . A system for identifying a plurality of nucleic acid molecules in a sample, said system comprising:
 (i) an inlet port;   (ii) an outlet port; and   (iii) a cartridge fluidically coupling said inlet port and said outlet port and defining a space containing:   a product capture housing enclosing a solid support with a plurality of product capture subdivisions configured in separate rows and columns, with each separate product capture subdivision comprising an array of a plurality of individual hydrophilic micro-pores, wherein each of said individual hydrophobic micro-pores is separated by hydrophobic surfaces each having opposed first and second open ends with the first end having a large diameter and the second end having a diameter which is smaller than that of the first end, said product capture housing comprising a plurality of fluid channels to permit material to pass from said inlet port through a column of the product capture subdivisions into contact with the array of micro-pores in those subdivisions, and to said outlet port, wherein the plurality of fluid channels are located above and below the solid support.   
     
     
         69 . The system of  claim 68  further comprising:
 one or more valves for selectively introducing or removing reagents and/or reactants into or out of said product capture housing through said inlet port or through said outlet port. 
 
     
     
         70 . The system of  claim 68  further comprising:
 one or more heating elements in said cartridge proximate to said product capture housing. 
 
     
     
         71 . A method for preparing a system for identifying a plurality of nucleic acid molecules in a sample, said method comprising:
 providing the system of  claim 68  and   applying capture oligonucleotide primers or probes to the micro-pores of the product capture subdivisions on the solid support within said product capture housing, whereby the capture oligonucleotide primers or probes are retained within the micro-pores or micro-wells.   
     
     
         72 . A process of identifying a plurality of nucleic acid molecules in a sample using the system prepared by the method of  claim 71 , wherein, following said applying capture oligonucleotide primers or probes to the micro-pores, said process comprises:
 conducting the reactions in said system and   detecting the presence of target nucleic acid molecules in the sample in the micro-pores based on said conducting the reactions.