Methods for producing polypeptide-tagged collections and capture systems containing the tagged polypeptides
First Claim
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1. A method for evenly distributing tags among members of a starting library, comprising:
- a) optionally adjusting the diversity of a starting library so that the diversity is within an order of magnitude of the number of molecules in the library;
b) dividing the starting library into “
n”
sublibraries designated 1 to n, wheren n is equal to or less than the number of unique tags, wherein each unique tag specifically binds to a different capture agent;
c) attaching a tag to a plurality of members of each sublibrary to produce “
n”
tagged sublibraries containing tagged members, wherein each member has the same tag, and the tag is unique to each sublibrary;
d) mixing some or all of the tagged sublibraries to produce a mixed library, wherein the number of tagged molecules added from each sublibrary is the same; and
e) splitting the mixed library into “
q”
array libraries, wherein q is from 1 up to a predetermined number of arrays.
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Abstract
Methods for evenly distributing tags on collections of molecules are provided. Also provided are assay methods that employ the tagged collections.
130 Citations
134 Claims
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1. A method for evenly distributing tags among members of a starting library, comprising:
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a) optionally adjusting the diversity of a starting library so that the diversity is within an order of magnitude of the number of molecules in the library;
b) dividing the starting library into “
n”
sublibraries designated 1 to n, wheren n is equal to or less than the number of unique tags, wherein each unique tag specifically binds to a different capture agent;
c) attaching a tag to a plurality of members of each sublibrary to produce “
n”
tagged sublibraries containing tagged members, wherein each member has the same tag, and the tag is unique to each sublibrary;
d) mixing some or all of the tagged sublibraries to produce a mixed library, wherein the number of tagged molecules added from each sublibrary is the same; and
e) splitting the mixed library into “
q”
array libraries, wherein q is from 1 up to a predetermined number of arrays. - View Dependent Claims (5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 41, 42, 45, 53, 54, 123)
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2. A method for evenly distributing nucleic acid molecules that encode polypeptide tags among members of a starting library, comprising:
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a) optionally, adjusting the diversity of a starting library so that the diversity is within an order of magnitude of the number of members in the library;
b) dividing the starting library into “
n”
sublibraries designated 1 to n, wherein n is equal to or less than the number of different nucleic acid molecules having nucleic acid molecules encoding different polypeptide tags;
c) attaching a nucleic acid molecule encoding a polypeptide tag to members of each sublibrary to produce “
n”
tagged sublibraries containing tagged members, wherein the encoded polypeptide tag is unique to each sublibrary;
d) mixing some or all of the tagged sublibraries to produce a mixed library, wherein the number of tagged nucleic acid molecules added from each sublibrary is the same;
e) splitting the mixed library into “
q”
array libraries, wherein q is from 1 to a predetermined number of arrays. - View Dependent Claims (3, 4, 8, 18, 19, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 121, 122, 124, 125, 126, 127, 128, 129, 131, 132)
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22. The method of 21, wherein the diversity is adjusted to be about equal to the number of molecules in the library.
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43. A capture system, comprising:
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an addressable collection of capture agents, wherein each locus in the collection contains capture agents that specifically bind to the same polypeptide tag, wherein the tags are evenly distributed among the tagged polypeptides;
a plurality of different polypeptide-tagged molecules bound to the capture agents, wherein the polypeptide-tagged molecules are sorted according to their specificity for the capture agents, wherein the tags are evenly distributed among the tagged molecules such that the diversity of tagged molecules at each locus in the collection is within one order of magnitude between and among loci.
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44. A capture system, comprising:
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an addressable collection of capture antibodies, wherein each locus in the collection contains antibodies that specifically bind to the same polypeptide tag;
a plurality of different polypeptide-tagged antibodies or fragments thereof bound to the capture antibodies;
wherein the polypeptide-tagged antibodies or fragments thereof are sorted according to their specificity for the capture antibodies; and
wherein the tags are evenly distributed among the tagged polypeptides such that the diversity of tagged molecules at each locus in the collection is within one order of magnitude.
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- 46. The capture system of 42, wherein each locus in the capture system further comprises an additional agent or plurality thereof at one or more loci, wherein the additional agents are common to a plurality of loci, and bind to and/or interact with captured biological particles and/or captured molecules.
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55. A collection of tagged molecules, wherein:
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the tags are evenly distributed among the tagged molecules such that the number of molecules having each tag is within 1.0, 0.5, 0.1, 0.05, or 0.01 order of magnitude; and
the collection has a diversity of at least 103. - View Dependent Claims (56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 133, 134)
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69. A method for capturing molecules, comprising:
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contacting a capture system with molecules under conditions whereby molecules bind to the capture system, wherein;
the capture system comprises a plurality of addressed loci;
the capture system comprises an addressed collection of polypeptide-tagged molecules bound to addressed capture agents at each locus;
the capture agents at each locus bind to the same polypeptide tag;
the polypeptide tag to which the capture agent binds is different among the loci;
each locus in capture system contains a plurality of different molecules each with the same tag bound to the capture agents; and
the polypeptide tags are evenly distributed among the tagged molecules such that the diversity of tagged molecules at each locus in the capture system is within one order of magnitude. - View Dependent Claims (70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118)
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119. A method of sorting molecules or reducing the diversity thereof, comprising:
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a) contacting a collection of tagged molecules with an array of addressed capture agents, wherein;
the agents at each addressed locus specifically bind the same tag, which differs from the tag to which agents at other loci bind;
the tags are evenly distributing among the tagged molecules; and
on the average, each tagged molecule is unique in each array library;
b) identifying from among the tagged molecules those having a predetermined activity or property;
c) based upon the tag(s) of the identified molecules, identifying the molecules linked to the tag, thereby sorting the molecules based upon the tag.
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120. A method of reducing the diversity of a collection of molecules, comprising:
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a) contacting a collection of tagged molecules with an array of addressed capture agents, wherein;
the agents at each addressed locus specifically bind the same tag, which differs from the tag to which agents at other loci bind;
the tags are evenly distributing among the tagged molecules; and
on the average, each tagged molecule is unique in each array library;
b) identifying from among the tagged molecules those having a predetermined activity or property;
c) based upon the tag(s) of the identified molecules, identifying the molecules linked to the tag;
d) selecting the molecules linked to the tag, thereby reducing the diversity of the collection of molecules.
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130. The method of 129, wherein the diversity is adjusted to be about equal to the number of molecules in the library.
Specification