Creation of functionalized microparticle libraries by oligonucleotide ligation or elongation
View Patent ↗Disclosed are methods of for constructing a bead-displayed library of oligonucleotide probes (or sequence-modified capture moieties such as protein-nucleic acid conjugates) by ligation of a capture probe, having an analyte-specific sequence, to an anchor probe that is attached, at its 5′-end, (or possibly at the 3′ end) to an encoded carrier such as a color-coded microparticle (“bead”). Such a library can also be constructed by elongation of an anchor probe, using a second probe as the elongation template, wherein the second probe has an anchor-specific subsequence and an analyte-specific subsequence.
1. A method of creating a carrier-displayed library of oligonucleotide probes from a carrier-displayed general purpose library of oligonucleotide probes in a single multiplex reaction by ligation of anchor probes attached to encoded carriers and analyte-specific capture probes, comprising:
a. providing a general purpose library of anchor oligonucleotide probes, wherein anchor probes having different sequences are attached to differently encoded carriers;
b. providing an analyte specific library of capture oligonucleotide probes which include subsequences complementary to designated analyte sequences;
c. providing a library of clamp probes, each member of the library having a unique sequence and a first subsequence complementary to a terminal subsequence of an anchor probe and a second subsequence complementary to a terminal subsequence of a capture probe such that for each of the clamp probes, their entire first subsequence participates in duplex formation with the complementary anchor oligonucleotide probes and their entire second subsequence participates in duplex formation with the capture oligonucleotide probes without any internal mismatches or terminal overhangs;
d. providing conditions permitting formation of tertiary complexes of capture probes, anchor probes and clamp probes;
e. incubating said tertiary complexes under conditions such that the capture and the anchor probe are ligated to each other to generate a single oligonucleotide, wherein no monomers are added during or after ligation, and
f. washing to release the clamp probes from the tertiary complexes.
2. The method of claim 1 wherein the carriers are microbeads and the encoding is with an optically detectable signal.
3. The method of claim 2 wherein the optically detectable signal is color.
4. A method of creating a carrier-displayed library of oligonucleotide capture probes in a single multiplex reaction by ligation of a first set of probes, including probes of differing sequences attached to encoded carriers, to a second set of probes having subsequences complementary to an analyte, by:
forming a complex using a third set of probes, each set having a unique sequence, capable of annealing to a first subsequence at or near the terminus of members of the first set and a second subsequence at or near the terminus of members of the second set wherein said third set of probes forms a duplex with both said first and second set; and
ligating said first set to said second set of probes, wherein no monomers are added during or after ligation.