Creation of Functionalized Microparticle Libraries by

Oligonucleotide Ligation or Elongation Related Applications

This application claims priority to US Provisional Application Serial No. 60/686333, filed 06/01/2005.

Background References (all incorporated by reference)

The following can be referred to as background in order to aid in understanding of certain of the terms and expressions below. "Oligo-Ligation via nick sealing"- Cherepanov, A.V. and de Vries, S. Kinetics and thermodynamics of nick sealing by T4 DNA ligase. Eur. J. Biochem., 270, 4315-4325 (2003).

"Chemical Ligation" - Xu, Y. and Kool, E.T. High sequence fidelity in a non- enzymatic DNA autoligation reaction. Nucleic Acids Research, 1999, vol. 27, no. 3, 875-881. "Ligation based SNP detection" - Dubiley, S. et al. Fractionation, phosphorylation and ligation on oligonucleotide microchips to enhance sequencing by hybridization. Nucleic Acids Research, 1997, vol. 25, No. 12, 2259-2265.

"Sequencing via cleavage and ligation" - Brenner, S. et al. In vitro cloning of complex mixtures of DNA on microbeads: Physical separation of differentially expressed cDNAs. Proc. Natl. Acad. Sci. USA, vol. 97, no. 4, 1665-1670 (2000).

Hermanson, G.T. 1996. Bioconjugate Techniques, Academic Press, San Diego, CA Liu, P., Burdzy, A., Sowers, L.C. "DNA ligases ensure fidelity by interrogating minor groove contacts" Nucleic Acids Res. 32, 15, 4503-4511 (2004). Broude, N.E., Sano, T., Smith, C.L., Cantor,C.R. "Enhanced DNA sequencing by hybridization" Proc. Natl. Acad. Sci. USA, Vol. 91, 3072-3076 (1994).

Iannone, M.A et al. "Multiplexed Single Nucleotide Polymorphism Genotyping by Oligonucleotide Ligation and flow cytometry", Cytometry 39 : 131-140 (2000). Gerry, N.P. et al. "Universal DNA microarray method for multiplex detection of low abundance point mutations", J. MoI. Biol. 292, 251 - 262 (1999). Background Discussion

The functionalization of solid phase carriers, notably microparticles ("beads"), for chemical and biological analysis, is generally is accomplished using a variety of

covalent conjugation chemistries, including the EDAC-mediated reaction (see Hermanson, G.T. 1996. Bioconjugate Techniques, Academic Press, San Diego, CA, incorporated by reference). Libraries of encoded functionalized microparticles for use in multiplexed formats of interrogation of nucleic acid sequence configurations — for example, those discussed in US Patent No. 6,797,524 and US Patent Application Serial No. 10/032,657, filed 12/28/2001 (both incorporated by reference) which use the Random Encoded Array Detection (READ™) format, where a bead array is formed on a substrate (a "BeadChip ™") - generally comprise a multiplicity of bead types, distinguishable, for example, by color, each type displaying one analyte-specific capture sequence. Analogous considerations apply to libraries of bead-displayed capture moieties ("receptors") for use in the multiplexed capture of proteins ("ligands").

Functionalization by covalent attachment requires the chemical modification of each analyte-specific capture probe, for example by animation of the 5 '-end using amine-modified dNTP's and appropriate linker moieties, for attachment to carrier- displayed carboxyl groups in the standard EDAC-mediated reaction (Hermanson G.T, supra). Oftentimes such immobilization protocols lead to improper orientation and steric hindrance problems, most of which can be removed by introduction of spacer molecules. While widely practiced, these chemical modifications nevertheless require special purification, usually by HPLC, and this purification step lowers the yield, raises the cost, and delays procurement. Further, each analyte-specific capture probe sequence is exposed to the not always gentle conditions of the attachment reaction which may introduce damage to the capture moiety, the degree of which may be difficult to assess. In addition, from a regulatory point of view, each modified carrier may be considered a separate reagent, requiring separate qualification.

From the point of view of manufacturing of encoded solid phase carriers, it will be beneficial to have a method of producing functionalized encoded microparticle libraries, where the microparticles bear a designated probe coverage {i.e., the number of probes/bead surface area) without the need for elaborate chemical modification of the analyte-specific capture probe sequences. Further, less chemical modification is more desirable from a regulatory point of view.

Summary of the Invention

The present invention discloses a method, for constructing a bead-displayed library of oligonucleotide probes (or sequence-modified capture moieties such as protein-nucleic acid conjugates, see US Application Serial No. 10/227,012, incorporated by reference) 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"). In one embodiment, an array of color-encoded microparticles configured in accordance with the READ™ format of multiplexed analysis (see US Patent No. 6,797,524, incorporated by reference) may be functionalized in a single on-chip reaction.

In another embodiment, a library of oligonucleotide probes (or sequence- modified capture moieties such as protein-nucleic acid conjugates, see US Application Serial No. 10/227,012) is generated by elongation of an anchor probe, using a template probe having an analyte-specific subsequence and an anchor probe-specific subsequence. The anchor probe is attached, at its 5 '-end, to an encoded carrier such as a color-coded microparticle ("bead"). The template probe is annealed through the anchor probe-specific subsequence, and the anchor probe is elongated with deoxynucleotide tri-phosphate (dNTPs) complementary to the analyte-specific subsequence to generate an elongation product, capable of capturing the analyte {see US Application Serial No. 10/271,602, for a description of the elongation process, incorporated by reference.) hi yet another embodiment, a library of bead bound oligonucleotide probes (or sequence-modified capture moieties such as protein-nucleic acid conjugates) is generated by either ligation or elongation as described above. However, in this case, the anchor sequence in addition to serving as an address sequence for the capture probe of interest is also utilized as a decode sequence. In this embodiment thus each bead type is defined by a combination of its fluorescent encoding as well as the unique anchor sequence attached to it that is recognized by a complementary decoder. The complementary decoder can be for example, a fluorescently labeled complementary oligonucleotide.

Brief Description of the Figures

Fig. 1 depicts the bead, with an anchor oligo attached and "clamped" to a capture oligo with a unique subsequence capable of hybridizing with a particular target.

Fig. 2A depicts generating a library of capture oligo functionalized beads using the ligation methods herein. Fig. 2B depicts generating a library of capture oligo functionalized beads using the ligation methods herein

Fig. 3 depicts the process of converting a BeadChip™ displaying anchor-oligo functionalized bead array to an analyte specific bead array using methods herein.

Fig. 4 depicts an in-solution ligation of probes 1 and 3 (with and without ligase), followed by attachment of the ligated product to a bead, removal of the clamp, and detection of the ligated product.

Fig. 5 depicts attachment of an anchor probe to a bead, followed by ligation of an analyte-specific probe 3, with and without ligase, removal of the clamp, and detection of the ligated product. Fig. 6 depicts a summary of an experiment demonstrating multiplexed ligation. The analyte-specific probes 3, 6 and 9 are shown to be ligated in a specific manner via hybridizations to their labeled respective targets 4, 7 and 10.

Fig. 7 depicts a summary of an experiment demonstrating that the hybridization performance of ligated and covalently coupled analyte specific probe are comparable. Fig. 9 shows making an encoded bead with a capture oligo with a unique subsequence capable of hybridizing with a particular target, using hybridization-mediated elongation rather than ligation.

Detailed Description

In one embodiment, encoded carriers are functionalized in two steps, namely: a first step of covalently attaching to a multiplicity of carriers an "anchor" probe having a sequence that generally is unrelated to any of the analyte-specific sequences; and a second step of ligating, to the anchor sequence on a given carrier type, a capture probe having an analyte-specific sequence that is recognizably associated with the carrier code. The step of ligating the two probes can be catalyzed by a DNA ligase. The ligation is achieved by using a third clamp-probe which has a sequence so as to allow hybridization of the anchor and capture probe immediately adjacent to each other. The

temporary hybridization complex in which anchor and capture sequences are annealed immediately adjacent to one another to the clamp, thereby form a duplex with a "nick" which can be sealed by a ligase-catalyzed formation of a phosphodiester bond between the 3' hydroxyl group of the anchor sequence and the 5' phosphate group of the capture sequence (Cherepanov, A. V. and de Vries, S. Kinetics and thermodynamics of nick sealing by T4 DNA ligase. Eur. J. Biochem., 270, 4315-4325 (2003)) to produce a single oligonucleotide (Fig. 1). hi another embodiment, chemical ligation also may be used (Chemical Ligation; Hermanson, G.T. 1996. Bioconjugate Techniques, Academic Press, San Diego, CA; Xu, Y. and Kool, E.T. High sequence fidelity in a non-enzymatic DNA autoligation reaction. Nucleic Acids Research, 1999, vol. 27, no. 3, 875-881). The use of this ligase-catalyzed reaction in several diagnostic applications, notably in a solid phase format, has been previously described. Examples include the reliable detection of a single-nucleotide polymorphism (SNP) or mutation at the site of the "nick" (Dubiley, S. et al., supra), microsequencing (Brenner, S. et al., supra). hi all such applications, DNA ligase is utilized to produce a covalent phosphodiester bond between the bead bound probe (carrying a 3' hydroxyl group) and the target oligonucleotide strand (carrying a 5' phosphate group). The methods rely on the fact that DNA ligases are sensitive to mis-paired nucleotides (mismatches) present on the 3' side of the ligated junction but somewhat tolerant of mismatches on the 5' side [Liu, P., Burdzy, A., Sowers, L.C. "DNA ligases ensure fidelity by interrogating minor groove contacts" Nucleic Acids Res. 32, 15, 4503-4511 (2004)]. This requirement that DNA ligases need fully base-paired duplexes near to the DNA junction has also been exploited to improve the performance of sequencing by hybridization [Broude, N.E., Sano, T., Smith, C.L., Cantor,C.R. "Enhanced DNA sequencing by hybridization" Proc. Natl. Acad. Sci. USA, Vol. 91, 3072-3076 (1994)] multiplexed SNP detection [Iannone, M.A et al. "Multiplexed Single Nucleotide Polymorphism Genotyping by Oligonucleotide Ligation and flow cytometry", Cytometry 39 : 131-140 (2000)] and for multiplexed detection of low abundance point mutations via a polymerase chain reaction/ligation detection reaction followed by hybridization to "zip-code" array [Gerry, N.P. et al. "Universal DNA microarray

method for multiplex detection of low abundance point mutations", J. MoI. Biol. (1999) 292, 251 - 262].

Herein, ligation is applied to a different purpose, namely the creation of a library of functionalized solid phase carriers, and especially of functionalized encoded microparticles ("beads").

In one embodiment of the method, some or all of the carrier types selected for the library may display the same anchor sequence. Thus, a set of carrier types displaying the same anchor sequence would constitute a general-purpose reagent which could be converted by the method of the invention to acquire analyte specificity. That is, by using clamps having sequences complementary to the same anchor sequence but different capture sequence, the same set of carrier types is readily functionalized in different ways. Further, two sublibraries containing carriers having identical color code, but displaying anchor probes of different sequence, may be mixed and decoded via interrogation of anchor sequences (that is, the anchor sequences function as an additional means by which to encode the array, which is decoded by annealing of the unique complementary sequence).

In another embodiment, this method also permits the creation, in a single-tube reaction (Fig. 2), of an entire library of carrier-displayed probe sequences, each carrier type within the library being associated with an anchor probe having a type-specific sequence. Similarly, an assembled planar array composed of multiple color-coded types of beads, each type displaying an anchor probe having a type-specific sequence {see Fig. 3) could be converted to an analyte-speciflc array via the ligation protocol disclosed herein. From a manufacturing point of view, this method would permit the order of array assembly and carrier functionalization to be reversed thereby permitting functionalization to be performed, either in a wafer-scale format, or in a chip-scale format, in a manner limiting the amount of reagent consumed. See US Application Serial No. 10/192,352, regarding wafer-scale and chip-scale assembly. From a regulatory point of view, the advantage of such an approach would be to be able to classify the assembled array as a general-purpose reagent. Specifically, the on-chip conversion of this general-purpose array into an application-specific array, calls for three reagents, two general-purpose

reagents, namely the array displaying the set of anchor probes and a set of clamps, and a set of analyte-specific reagents, namely the capture probes. The assembled array will be composed of multiple types of beads with type-specific anchor sequences. Clamps will have one subsequence that is complementary to an anchor-sequence and one subsequence that is complementary a portion of the sequence of the capture probe. The third reagent namely the capture probe, will be an analyte-specific reagent (ASR). In one embodiment, the capture probe will have a portion that interacts with the target and another portion that interacts with the clamp.

In another embodiment, the on-chip conversion of the general-purpose array into an application-specific array is performed not by ligation but by a polymerase- catalyzed elongation reaction using the analog of a clamp sequence as an elongation template. That is (see Fig. 8), as before, the 3 '-end of the clamp sequence is designed to form a duplex with a specific anchor sequence displayed on an encoded microparticle under conditions permitting the elongation of the anchor sequence in a manner described in US Application Serial No. 10/271,602 (incorporated by reference), but not necessarily with incorporation of fiuorescently, tagged dNTPs pr ddNTPs. The "overhang" of the template, typically 10-30 nt and more typically 15-25 nt in length, is selected to be identical to a specific target sequence of interest. Elongation adds to the 3 'end of the anchor sequence an application-specific subsequence that is complementary to the 5 '-overhang of the template sequence {see US Application Serial No. 10/271,602). Following completion of the elongation reaction, the template and reaction constituents are removed (for example by washing in water, at elevated temperature). In contrast to the ligation-catalyzed method, no capture probe sequence is needed, a fact that simplifies the design of "orthogonal" sequence sets (see also Example 8). Multiple elongation reactions can be performed in parallel, permitting the concurrent modification of an entire set of arrays. The availability of a general-purpose bead array permits a mode of co-development. Manufacturing generally will require facilities which will not be available to prospective users of the array who might wish to provide their own array composition for specific applications. The methods described herein provide for the separation of the step of array manufacturing - including the synthesis of a general-purpose library

of encoded beads, the assembly of bead arrays and their packaging into desired configurations - from the step of rendering the array application specific. The methods of array modification disclosed herein thus facilitate a modality of co-development by which the array manufacturer provides a set of reagents including the general-purpose array, as well as application-specific reagents such as the template sequence, in the case of the elongation-mediated method of the invention, and application developers produce application-specific arrays "as-needed." The array manufacturer thereby can engage in strategic co-development projects and collaborations while retaining control over technical know-how, while enabling the application developer to likewise retain control of sensitive information and permitting "on-site" experimentation with different sequences in a prototyping mode requiring rapid turn-around. The methods of the invention thereby permit the array manufacturer to extend the range of application of pre-assembled arrays. Examples

Example 1 Synthesis and design of oligonucleotide sequences

All oligonucleotide synthesis was carried out by IDT (Coralville, IA). The sequence information and the end modifications are shown in Table 1. Table 1

Oligo# Olgio Name Sequence (5'- 3')

1 DR-70e-Biotin Biotin-TEG-CAG AAG GAC ATC CTG GAA G (SEQ E ⁾ NO: 1

2 LK-70e-HA114 TCG GTC TTC CAG GAT (SEQ E) NO: 2

3 P-HAl 14 PO ₄ ^"" - ACC GAG AGA GCC TGC GGA T (SEQ TD NO: 3

4 T-HAl 14 Cy3 - ATC CGC AGG CTC TCT CGG T (SEQ E ⁾ NO: 4]

5 LK-70e-HA107 TCC TGC TTC CAG GAT (SEQ E ⁾ NO: 5

6 P-HA107 P(V-CAG GAG AGG CCT GAG TAT T (SEQ E) NO: 6]

7 T-HAl 07 Cy3 - AAT ACT CAG GCC TCT CCT G (SEQ E) NO: T

8 LK-70e-HA125 CGC CTC TTC CAG GAT (SEQ E) NO: 8

9 P-HA125A PCV-AGG CGG TCC ATG CGG C (SEQ E ⁾ NO: 9

Example 2: In-solution ligation followed by capture of ligated product on bead

For this example oligos 1, 2 and 3 were used. Hybridization was performed by mixing 5ul aliquots of lOuM oligo 1,2 and 3 in solution, heating the solution to 95°C, holding for five minutes and slowly cooling it back to room temperature. Ligation was carried out at RT for 2 hours by taking 5 ul of the hybridization mix and adding to it 2ul of 10x T4 Ligase Reaction buffer (supplier info) and IuI of T4 ligase enzyme. The reaction volume was made to 20ul by addition of 12 ul of deionized molecular biology grade water. The biotinylated reaction product was captured using color-encoded Neutravidin functionalized beads. 1OuI of 1% beads was added to 20ul of the ligation reaction product and the reaction volume adjusted to 350ul by adding IxPBS. The suspension was incubated at room temperature for 30 minutes with shaking. Following capture reactions, the beads were washed 2x with PBST and re-suspended in lOul PBS. The beads were then assembled, on chip, and the hybridized duplex was disrupted and the clamp-oligo removed via a stringent wash at 53 ⁰ C (wash buffer:m20mM TRIS, pH 7.5, O.lx SSC, 0.01% SDS). Finally the detection of the ligated product was carried out via hybridization by using a series of dilutions of the labeled probe 4 in IxTMAC. The hybridization was carried out using 20ul of solution/chip, at 53 ⁰ C for 25 minutes. Post hybridization washing was performed using 0.7xTMAC and at 53 ⁰ C. Finally the CMp was mounted on to the BAS AIS system and the fluorescent images recorded. The results are shown in Figure 4. Example 3 On-bead ligation

The experiment was identical to Example 1, except the anchor sequence (#1) was immobilized on a Neutravidin functionalized color encoded bead first. The ligation was hence carried out using the ternary hybridization complex (oligos 1+2+3) tethered to the bead. The results are shown in Figure 5.

Example 4 Multiplexed on bead ligation

Two pairs of particle captured ternary hybridization duplexes were produced using methods outlined in Example 1 and 2. Pair 1 : oligos (1+2+3) and oligos(l+5+6) and Pair 2 : (1+2+3) and (1+8+9) (each prepared separately).

On-bead ligation reaction was performed as disclosed above followed by stripping off clamp oligos 2 and 5 (pair 1) and oligos 2 and 8 (pair 2). Finally the presence and specificity of the ligated probes was checked via separate hybridizations with labeled targets 7 and 4 (pairl) and labeled targets 10 and 4 (pair 2). The results are shown in Figure 6.

Example 5 Comparison of chemically coupled and ligated analyte-specific capture probes

5' animated probes 11 and 12 were coupled to two different color encoded particles using an EDAC reaction under nominally identical conditions. The bead coupled with probe 12 was subjected to ligation using clamp and capture oligos 2 and 3, respectively. The bead coupled with probe 12 was directly used for target (No. 4) detection via hybridization. The results are shown in Figure 7. Example 6 Oligonucleotide design for multiplex on-chip ligation

A number of different computer programs are available for design of oligonucleotides to be used as PCR primers, molecular beacons or other applications. For each application a different set of constraints need to be imposed on the design process. No existing software, to the best of our knowledge, provides designs or constraints suitable for use with the ligation or elongation products described herein. This example describes the design process for a set of anchor, clamp and capture probes for use in multiplexed on-chip ligation.

The function of the ss-anchor oligo is to interact with its perfect complement (clamp) and not to bind any other oligo in solution. A significant amount of scientific literature is available on the design algorithms of such non-interacting tag sequences (Gerry, N.P. et al. Universal DNA microarray method for multiplex detection of low abundance point mutations. J. MoI. Biol. (1999) 292, 251-262; Liu, Q. et al. DNA computing on surfaces. Nature (2000) 403,175-178). For the current study we chose

to use two sets of published tag sequences. The first set (30 sequences) was obtained from the genome website at Massachusetts Institute of Technology. The tags were 25- 27 bases in length, T _m ~ 55 ⁰ C at a salt cone, of 5OmM and contained no more than two consecutive identical bases. The tags were checked using BLAST and found non homologous to all known human genes. The second set (13 sequences) was collected from a published set of GeneFlex Tag Array sequence collection (Affymetrix, Santa Clara, CA) that contains sequence information for 2000 oligonucleotides with minimal tendency for cross-hybridization. The sequences were 20 bases long.

The function of the ss-capture oligo is to interact with the cognate target and the clamp sequence and not bind any other oligo in solution. The choice of the capture sequence is determined by the assay of interest. For this example a set of probes for the HLA DQ locus was selected, As described above, since the sequence tags are unrelated to the particular set of probes (targeting a human gene) in question the approach is generic and can be used for any set. Once a potential pool of anchor sequences and a set of capture sequences were chosen (see Table 3), the anchor sequences were checked for their 3' similarity. The sequences which had 3 'similarity were identified and flagged (see Table 4). Next the capture sequences were checked for their similarity in the first 5 bases from the 5' end. If found, similar additional bases were inserted at the 5' end and the check repeated, until all the 21 probes had unique 5' ends. The process left the starting capture sequences minimally perturbed and a maximum of two additional 5' base insertions were needed (see Table 5). Finally the set of all the tag sequences were checked for homology with the probe sequences and their reverse complements. Table 3 List of anchor probe and capture probe sequences

List of anchor probe sequences

Bl CGCAGGTATCGTATTAATTGATCTGC (SEQ IDNO: 13) Al GTTGATGTCATGTGTCGCAC (SEQ ID NO:

B2 CCTCATGTCAACGAAGAACAGAACC (SEQIDNO: 14) A2 TCGTGCCTTGTCATTCGGGA (SEQID NO:

B3 ATTGAAGCCTGCCGTCGGAGACTAA (SEQIDNO: 15) A3 CGTGCAAGTTACCGAGCTGA (SEQE) NO:

B4 AGΆCTGCGTGTTGGCTCTGTCACAG (SEQ IDNO: 16) A4 TAGATCAGTTGGACTCGATG (SEQ ID NO:

B5 TTATGGTGATCAGTCAACCACCAGG (SEQIDNO: 17) A5 GCAGGGAATTGCCGACCATA (SEQID NO:

B6 GAGACACCTTATGTTCTATACATGC (SEQIDNO: 18) A6 ACGTTCGTCAAGAGTCGCAT (SEQ ID NO:

B7 TCCATGCGCTTGCTCTTCATCTAGC (SEQIDNO: 19) A7 CGTTCCTAAAGCTGAGTCTG (SEQIDNO:

B8 GCCTTACATACATCTGTCGGTTGTA (SEQ IDNO: 20) A8 GAGAGGCCGTCGCTATACAT (SEQID NO:

B9 CACAAGGAGGTCAGACCAGATTGAA (SEQ IDNO: 21) A.9 AAGCCAGATCGACCATCGTA (SEQ ID NO:

BlO GCCACAGATAATATTCACATCGTGT (SEQ ID NO: 22) AlO GACGCCGTTATGAGAGTCCA (SEQIDNO:

BU ACACATACGATTCTGCGAACTTCAA (SEQ ID NO: 23) All ATATCGTGTCACAGGTCGTT (SEQ ID NO:

B12 TTACAGGATGTGCTCAACAGACGTT (SEQ ID NO: 24) -U2 ATGATGTGCAAAGTGCCGTC (SEQIDNO:

B13 GCTCACAATAATTGCATGAGTTGCC (SEQ ID NO: 25) A13 TCCGTCTGTTGAGTTAGGCC (SEQIDNO:

B14 CTGCACTGCTCATTAATATACTTCTGG (SEQ ID NO: 26) A14 CTCGACCGTTAGCAGCATGA (SEQIDNO:

B15 TTCACGCACTGACTGACAGACTGCTT (SEQ ID NO: 27) Al5 CCGAGATGTACCGCTATCGT (SEQIDNO:

B16 CAACATCATCACGCAGAGCATCATT (SEQ ID NO: 28) A16 AGAGCGCATGAATCCGTAGT (SEQIDNO:

B17 GCATCAGCTAACTCCTTCGTGTATT (SEQ ID NO: 29)

B18 GGCGTTATCACGGTAATGATTAACAGC (SEQ ID NO: 30)

B19 ACATCAATCTCTCTGACCGTTCCGC (SEQ ED NO: 31)

B20 GCCTTATGCTCGAACTGACCATAAC (SEQ ID NO: 32) B21 CGSATATCACCACGATCAATCATAGGTAA (SEQ ID NO: 33)

B22 CCTTAATCTGCTGCAATGCCACAGC (SEQ ID NO: 34)

B23 TAGCTCTCCGCCTACAATGACGTCA (SEQ ID NO: 35)

B24 AGGAACGCCTTACGTTGATTATTGA (SEQ ID NO: 36)

B25 GAGTCAGTACCGATGTAGCCGATAA (SEQ ID NO: 37)

B26 ACTCGAATGAACCAGGCGATAATGG (SEQ ID NO: 38)

B27 ATTATATCTGCCGCGAAGGTACGCC (SEQ ID NO: 39)

B28 GGACAGACAGTGGCTACGGCTCAGTT (SEQ ID NO: 40)

B29 CGGTATTCGCTTAATTCAGCACAAC (SEQ ID NO: 41)

B30 GCTCTTACCTGTTGTGCAGATATAA (SEQ ID NO: 42)

List of HLA DQ capture probe sequences

DQlOl CGGGGTGTGACCAGACACA (SEQ IDNO: 59) DQlIl CGTCTTGTAACCAGATACA (SEQ ID NO: 6 ¹ DQ102 GGGTGTACCGGGCAGTGAC (SEQ IDNO: 60) DQ112 CGTCTTGTGAGCAGAAGCA (SEQ ID NO.7( DQ103 GCGGCCTAGCGCCGAGTAC (SEQ IDNO: 61) DQ113 CGACGTGGAGGTGTACCGG (SEQ ID NO: 7 DQ104 GCGGCCTGTTGCCGAGT (SEQ IDNO: 62) DQH4 GccGccTGACGccGAGT (SEQ ID NO: 7: DQlO5 CGTTATGTGACCAGATACA (SEQ IDNO: 63) DQ115 GGCCGCCTGCCGCCGAGT (SEQ ID NO: T. DQ106 CGTCTTGTAACCAGACACA (SEQ IDNO: 64) DQIlS GACCGAGCGCGTGCGGGGT (SEQ ID NO: 7< DQ107 CGTCTTGTGACCAGATACA (SEQ IDNO: 65) DQ117 GCTGGGGCGGCTTGACGCC (SEQ ID NO: 7: DQ108 GCGGCCTGATGCCGAGTAC (SEQ IDNO: 66) DQ118 GGGTGTATCGGGCGGTGAC (SEQ ID NO: 7( DQl09 GACCCGAGCGGAGTTGGAC (SEQ IDNO: 67) DQ119 GGCGGCCTGACGCCGAGT (SEQ ID NO: T DQIlO GAGGGGACCCGGGCGGAGT (SEQ IDNO: 68) DQ120 CGCTTCGACAGCGACGTGG (SEQ ID NO: 7! DQ121 AACCGAGAAGAGTACGTGC (SEQ ID NO: 7!

Table4 Pairs ofanchorprobes showing 3' similarity

Al GTTGATGTCATGTGTCGCAC (SEQ ID NO: 80)

A6 ACGTTCGTCAAGAGTCGCAT (SEQ ID NO: 81) ^'

B25 GAGTCAGTACCGATGTAGCCGATAA (SEQ ID NO: 82)

B30 GCTCTTACCTGTTGTGCAGATATAA (SEQ ID WO: 83)

B18 GGCGTTATCACGGTAATGATTAACAGC (SEQ ID NO: 84)

B22 CCTTAATCTGCTGCAATGCCACAGC (SEQ ID NO: 85)

A14 CTCGACCGTTAGCAGCATGA (SEQ ID NO: 86)

B6 GAGACACGTTATGTTCTATACATGC (SEQ ID NO: 87)

Bl CGCAGGTATCGTATTAATTGATCTGC (SEQ ID NO: 88)

B14 CTGCACTGCTCATTAATATACTTCTGG (SEQ ID NO: 89)

All ATATCGTGTCACAGGTCGTT (SEQ ID NO: 90)

B12 TTACAGGATGTGCTCAACAGACGTT (SEQ ID NO: 91)

Table 5 Revised capture probe sequences withunique 5' ends

DQ101 CGGGGTGTGACCAGACACA (SEQ ID NO: 92)

DQ102 TTGGGTGTACCGGGCAGTGAC (SEQ ID NO: 93)

DQ103 TAGCGGCCTAGCGCCGAGTAC (SEQ ID NO: 94)

DQ104 TTGCGGCCTGTTGCCGAGT (SEQ ID NO: 95)

DQ105 CGTTATGTGACCAGATACA (SEQ ID NO: 96)

DQ10S TACGTCTTGTAACCAGACACA (SEQ ID NO: 97)

DQ107 TTCGTCTTGTGACCAGATACA (SEQ ID NO: 98)

DQ108 TCGCGGCCTGATGCCGAGTAC (SEQ ID NO: 99)

DQ109 GACCCGAGCGGAGTTGGAC (SEQ ID NO: 100)

DQ110 AGAGGGGACCCGGGCGGAGT (SEQ ID NO: 101)

DQ111 TCCGTCTTGTAACCAGATACA (SEQ ID NO: 102)

DQ112 GGCGTCTTGTGAGCAGAAGCA (SEQ ID NO: 103)

DQ113 CGACGTGGAGGTGTACCGG (SEQ ID NO: 104)

DQ114 CAGCCGCCTGACGCCGAGT (SEQ ID NO: 105)

DQ115 GGCCGCCTGCCGCCGAGT (SEQ ID NO: 106)

DQ116 TAGACCGAGCGCGTGCGGGGT (SEQ ID NO: 107)

DQ117 GCTGGGGCGGCTTGACGCC (SEQ ID NO: 108)

DQ118 TAGGGTGTATCGGGCGGTGAC (SEQ ID NO: 109)

DQ119 ATGGCGGCCTGACGCCGAGT (SEQ ID NO: 110)

DQ120 CGCTTCGACAGCGACGTGG (SEQ ID NO: 111)

DQ121 AACCGAGAAGAGTACGTGC (SEQ ID NO: 112)

Example 7 Multiplex on-chip ligation

Table 6 below summarizes the 5-probe system chosen for the multiplexed experiment. The table lists the individual sets consisting of the capture, clamp and the anchor probe and also shows their alignment.

Table 6 : Sequences for the multiplexed ligation experiment

DQl07CAPT

Phosphate-TTC GTC TTG TGA CCA GAT ACA (SEQ ID NO: 113)

DQl07ANCH

Amine-GTT GAT GTC ATG TGT CGC AC (SEQ ID NO: 114)

DQl07CLMP

ACG AAG TGC GAC ACA (SEQ ID NO: 115)

5'-gtt gat gtc atg tgt cgc ac ttc gte ttg tga cca gat aca-3' (SEQ ID NO: 116) ac aca gcg tg aag ca (SEQ ID NO: 117)

DQl08CAPT

Phosphate-TCG CGG CCT GAT GCC GAG TAC (SEQ ID NO: 118)

DQl08ANCH

Amine-CGT TCC TAA AGC TGA GTC TG (SEQ ID NO: 119)

DQl08CLMP

CGC GAC AGA CTC AGC (SEQ ID NO: 120)

5'-cgt tec taa age tga gtc tg teg egg cct gat gcc gag tac-3' (SEQ ID NO: 121) eg act cag ac age gc (SEQ ID NO: 122)

DQ11OCAPT

Phosphate-AGA GGG GAC CCG GGC GGA GT (SEQ ID NO: 123)

DQ11OANCH

Amine-AAG CCA GAT CGA CCA TCG TA (SEQ ID NO: 124)

DQ110CLMP

CCT CTT ACG ATG GTC (SEQ ID NO: 125)

5'-aag cca gat cga cca teg ta aga ggg gac ccg ggc gga gt-3' (SEQ ID NO: 126) ct ggt age at tct CC (SEQ ID NO: 127)

DQ112CAPT

Phosphate-GGC GTC TTG TGA GCA GAA GCA (SEQ ID NO: 128)

DQ112ANCH

Amine-ATG ATG TGC AAA GTG CCG TC (SEQ ID NO: 129)

DQ112CLMP

ACG CCG ACG GCA CTT (SEQ ID NO: 130)

5' -atg atg tgc aaa gtg ccg tc ggc gtc ttg tga gca gaa gca-3' (SEQ ID NO: 131) tt cac ggc ag ccg ca (SEQ ID NO: 132)

DQ120CAPT

Phosphate-CGC TTC GAC AGC GAC GTG G (SEQ ID NO: 133)

DQ120ANCH

Amine-AGA GCG CAT GAA TCC GTA GT (SEQ ID NO: 134)

DQ120CLMP

AAG GCG ACT ACG GAT (SEQ ID NO: 135)

5'-aga gcg cat gaa tec gta gt cgc ttc gac age gac gtg g-3' (SEQ ID NO: 136) tt agg cat ca gcg aa (SEQ ID NO: 137)

The experiment was carried out as follows: Step 1 : coupling of animated anchor probes to the beads:

The aminated anchor probes (DQ107ANCH, DQ108ANCH, DQIlOANCH, DQ112ANCH and DQ120ANCH) and one negative control probe (Nl 8) were covalently attached to six different color encoded microparticles using EDAC chemistry. They were then used to manufacture several BAS BeadChips. Step 2: Annealing of capture probe and clamps:

The following mixtures were made in five different eppendorf tubes and incubated at 94 ⁰ C for 10 minutes followed by cooling to room temperature.

Step 3: Preparation of the ligation master mix:

The ligation reaction mix was prepared by mixing together i) 1 OX Ligation Reaction buffer: 2ul (New England BioLabs, Ipswich, MA) ii) T4 Ligase enzyme: lul (New England BioLabs, Ipswich, MA) iii) 3ul of each of the annealed product from step 2 (total 15ul) iv) 2ul of DI water

Step 4: On-chip ligation:

The ligation mix (2OuI) prepared in step 3 was added to a BeadChip prepared in step 1 and incubated at RT for 1.5 hr in a humid chamber. Following this the chip was washed thoroughly at room temperature with DI water to strip off the clamp and any un- ligated capture probe. The chip was dried and stored at 4 ⁰ C until further use. Step 5: On-chip Hybridization:

Five oligos (reverse complements of the capture probes listed in Table 6) with a 5' biotin tag were used as the hybridization targets. IuM solutions of each were prepared using Ix TMAC solution. A pooled target mixture was also prepared using IxTMAC and all the five targets (Final target cone. IuM). 20 ul of each target solution was aliquoted onto a separate chip and incubated at 53 ⁰ C for 15 minutes. The sample was aspirated off and the chips were then washed with 20ul IxSSC buffer with 0.1%

SDS at 53 ⁰ C for 10 min. Following this the chips were stained with a 1:200 Streptavidin-CY3 solution and washed with 20ul IxSSC buffer with 0.1% SDS at RT for 5 min. The slide was then fixed with a fixative solution and finally rinsed with a stop solution and dried. The fluorescent signals were read using a BAS AIS system. The results are shown in Table 7 below. Except DQ 108 capture probe (which showed no signal), all other probes performed in a satisfactory fashion.

Table 7 : results of multiplexed on-chip ligation

Example 8: Design of multiplex on-chip elongation

This preferred embodiment requires a unique set of anchor sequences (each such sequence uniquely associated to an optically decodable code, such as a fluorescently encoded microparticle) and a matched template sequence, that is, a template sequence with a 3'-terminal subsequence that is complementary to the anchor sequence and a 5'-terminal subsequence that is identical to a selected subsequence within a target sequence, for example, a target subsequence comprising a designated variable site of interest. The tag-sequences discussed below in Example 6, with no mutual cross-reactivity and minimal homology to the human genome can be utilized for the anchor sequences of interest. Once the anchor sequences and the target subsequences of interest have been identified, the template sequence is constructed as described above. The process of array modification involves (see Figure 8) contacting a pool of the template oligos with an array of microparticles pre- functionalized with the anchor oligos in presence of dNTPs or ddNTPs and a DNA- polymerase enzyme in a suitable buffer. The polymerase extends the 3' end of the anchor sequences in matched anchor-template duplexes in accordance with the 5' overhang of the template sequence. Finally, the template sequence is de-hybridized

leaving behind the newly synthesized ss-oligo, which can now function as the capture probe of interest. It is worthwhile to mention that this particular approach of array modification requires only one unmodified template sequence to be pre-synthesized. It should be understood that the terms, expressions and examples herein are exemplary only and not limiting, and that the scope of the invention is defined only in the claims which follow, and includes all equivalents of the subject matter of those claims.