Address/capture tags for flow-cytometery based minisequencing

• US 20030190609A1
• Filed: 06/07/2001
• Published: 10/09/2003
• Est. Priority Date: 06/08/2000
• Status: Abandoned Application

• Current Assignee: David C. Torney, White P. Scott
• Original Assignee: David C. Torney, White P. Scott
• Inventors: White, P. Scott, Torney, David C.
• Application Number:
  US09/877,819
• Publication Number:
  US 20030190609A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/626 being a flow cytometer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

A method for generating address/capture tags for use in a sensitive and rapid flow-cytometry based assay for the multiplexed analysis of SNPs based on polymerase-mediated primer extension using microspheres as solid supports is described. Single-nucleotide polymorphisms (SNPs) are the most abundant type of human genetic variation. These variable sites are present at high density in the genome, making them powerful tools for mapping and diagnosing disease-related alleles. Subnanomolar concentrations of sample in small volumes (10 ml) can be analyzed at rates greater than one sample per minute, without a wash step. Genomic analysis using multiplexing microsphere arrays, enables the simultaneous analysis of dozens, and potentially hundreds of SNPs per sample. The method has been tested by genotyping the Glu69 variant from the HLA DPB1 locus, a SNP associated with chronic beryllium disease, as well as HLA DPA1 alleles.

Claims

1. A method for identifying a set of sequences useful as address/capture tags which comprises the steps of:

(a) generating a chosen number of single-stranded, random oligonucleotide sequences having a chosen length;

(b) rejecting all sequences from said chosen number of single-stranded, random oligonucleotide sequences having common subsequences with a subsequence length greater than a chosen number of bases, the remaining sequences forming a first group of sequences;

(c) rejecting all sequences in said first group of sequences which can form stable hairpins, the remaining sequences forming a second group of sequences; and

(d) rejecting all sequences in said second group of sequences which can form stable dimers, the remaining sequences forming a third group of sequences;

whereby a set of sequences is identified such that the sequences, if synthesized, would hybridize to their respective complements with a high degree of specificity.

2. The method for identifying a set of sequences useful as address/capture tags as described in claim 1, further comprising the step rejecting all reverse complementary sequences from said third group of sequences, the remaining sequences forming a fourth group of sequences;

3. The method for identifying a set of sequences useful as address/capture tags as described in claim 2, further comprising the steps of determining the melting temperature of each of sequence in said fourth group of sequences;

and rejecting all sequences that melt below a selected temperature, forming thereby a fifth group of sequences.

4. The method as described in claim 3, further comprising the steps of synthesizing a desired number of the sequences in the fifth group of sequences, and synthesizing the complements thereof.

5. The method for generating a set of address/capture tags as described in claim 3, wherein said selected melting temperature is between 50°

C. and 70°

C.

6. The method for generating a set of address/capture tags as described in claim 5, wherein said selected melting temperature is about 60°

C.

7. The method for generating a set of address/capture tags as described in claim 1, further comprising the step of rejecting all runs of bases greater than a chosen number of bases.

8. The method for generating a set of address/capture tags as described in claim 7, wherein the chosen number of bases is 2.

9. The method for generating a set of address/capture tags as described in claim 1, wherein said chosen number of random DNA sequences is computationally generated.

10. The method for generating a set of address/capture tags as described in claim 4, wherein said synthesized sequences are immobilized on identifiable microparticles, each of said synthesized sequences being immobilized on a different identifiable microsphere.

11. The method for generating a set of address/capture tags as described in claim 4, wherein said synthesized complementary sequences are immobilized on identifiable microparticles, each of said synthesized complementary sequences being immobilized on a different identifiable microsphere.

12. The method for generating a set of address/capture tags as described in claim 4, wherein the address/capture tags are used for multiplexed SNP scoring in a flow cytometer assay.

13. A method for generating a set of address/capture tags which comprises the steps of:

(a) generating a chosen number of single-stranded, random oligonucleotide sequences having a chosen length;

(b) rejecting all reverse complementary sequences from said chosen number of random oligonucleotide sequences, the remaining sequences forming a first group of sequences;

(c) rejecting all sequences having runs of bases greater than a chosen number of bases, the remaining bases forming a second group of bases;

(d) rejecting all sequences from said second group of sequences having common subsequences with a subsequence length greater than a chosen number of bases, the remaining sequences forming a third group of sequences;

(e) rejecting all sequences in said third group of sequences which can form stable hairpins, the remaining sequences forming a fourth group of sequences;

(f) rejecting all sequences in said fourth group of sequences which can form stable dimers, the remaining sequences forming a fifth group of sequences;

(g) determining the melting temperature of each of sequence in said fifth group of sequences;

(h) rejecting all sequences that melt below a selected temperature, forming thereby a sixth group of sequences;

(i) synthesizing a desired number of the sequences in said sixth group of sequences; and

(j) synthesizing the complementary sequences of said desired number of sequences, whereby a set of address/capture tags is generated such that the synthesized sequences hybridize to their respective complementary sequences with a high degree of specificity.