MULTIPLEXING IN PARTITIONS USING PRIMER PARTICLES
Described herein are microparticles each comprising a plurality of bound biological molecules. Further described herein is a plurality of microdroplets each comprising one or more primer vehicles. Methods of making and using these microdroplets are also reported. An exemplary microparticle is of Formula (I).
1 . A microparticle of Formula (I):
wherein
is a biological molecule;
m is an integer of 0 to 100, inclusive;
n is an integer of 0 to 100, inclusive;
R1 is a linker selected from the group consisting of a bond, optionally substituted alkylene, optionally substituted heteroalkylene, optionally substituted alkenylene, optionally substituted heteroalkenylene, optionally substituted alkynylene, optionally substituted heteroalkynylene, optionally substituted heterocyclylene, or optionally substituted heteroarylene, and
each of R2 and R3 is independently hydrogen, substituted or unsubstituted alkyl, or a nitrogen protecting group.
2 . The microparticle of claim 1 , wherein the microparticle is of Formula (II):
wherein R4 is optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heterocyclylene, or optionally substituted heteroarylene.
3 . The microparticle of claim 2 , wherein the microparticle is of Formula (II-a):
wherein p is an integer of 1 to 5, inclusive.
4 . The microparticle of claim 1 , wherein the microparticle is of Formula (II-b):
5 - 35 . (canceled)
36 . A plurality of microdroplets, each comprising a nucleic acid template molecule and a plurality of primer vehicles, wherein each primer vehicle comprises a plurality of primer species bound to a microparticle through a plurality of binding moieties, wherein each primer species is specific for a different target site of a nucleic acid template molecule.
37 - 38 . (canceled)
39 . The plurality of microdroplets of claim 36 , wherein at least one primer species is specific for a target site on the nucleic acid template molecule in at least one microdroplet.
40 - 43 (canceled)
44 . The plurality of microdroplets of claim 36 , wherein each of the binding moieties comprises a sequence complementary to a member of the primer pairs.
45 - 50 . (canceled)
51 . The plurality of microdroplets of claim 36 , wherein each of the primer vehicles has a single primer species bound.
52 . (canceled)
53 . The plurality of microdroplets according to any one of the preceding claims of claim 36 , wherein each of the primer vehicles has different primer species bound.
54 - 55 . (canceled)
56 . The plurality of microdroplets claim 36 , wherein each microdroplet has a plurality of same primer vehicles.
57 - 58 . (canceled)
59 . The plurality of microdroplets of claim 36 , wherein each primer species is a primer pair.
60 - 64 . (canceled)
65 . The plurality of microdroplets of claim 36 , wherein the primer species comprises a barcode, a universal sequence, and a target specific sequence.
66 - 71 . (canceled)
72 . A method for detecting a nucleic acid template molecule in a biological sample, comprising the steps of:
forming a plurality of microdroplets of any one of the preceding claims;
amplifying at least one nucleic acid template molecule in the microdroplets to give an amplified product; and
sequencing the amplified product.
73 . The method of claim 72 , wherein said forming step further comprises:
providing a first solution comprising a nucleic acid template molecule;
providing a second solution comprising a plurality of different primer species each specific for a different target site on a nucleic acid template;
merging the first and second solution to form a merged solution; and
partitioning the merged solution in an immiscible fluid.
74 . The method of claim 72 , before the sequencing step, further comprising merging one of the microdroplets with a microdroplet comprising a barcode.
75 - 78 . (canceled)