MULTIPLEXED GENOMIC GAIN AND LOSS ASSAYS

• US 20090148841A1
• Filed: 03/26/2008
• Published: 06/11/2009
• Est. Priority Date: 12/05/2007
• Status: Active Grant

• Current Assignee: Revvity Health Sciences Incorporated (Revvity, Inc.)
• Original Assignee: PerkinElmer LAS, Inc. (Perkinelmer Incorporated)
• Inventors: Schermer, Mack J., Adler, Karl E.
• Granted Patent: US 7,932,037 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2539/115 Comparative genomic hybridisation [CGH]
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Encoded bead multiplex assays for chromosomal gains and losses are provided that provide the benefits of complex, large template DNA sources, such as BAC DNA, as the probe material without bead networking or other assay performance problems. Reagents for assaying DNA are described herein which include a plurality of encoded particles having attached amplicons amplified from a template DNA sequence. Each individual attached amplicon includes a nucleic acid sequence identical to a random portion of the template DNA sequence, wherein the amplicons together represent substantially the entire template DNA and wherein the nucleic acid sequence identical to a random portion of the template DNA sequence of each individual amplicon is shorter than the entire template DNA.

Claims

1. A method of assaying a DNA sample, comprising:

providing a first encoded particle set comprising encoded particles having attached amplicons, the amplicons comprising random nucleic acid sequences together representing substantially an entire first template DNA sequence;

hybridizing the amplicons of the first encoded particle set with detectably labeled sample DNA;

hybridizing the amplicons of the first encoded particle set with detectably labeled reference DNA;

detecting a first signal indicating specific hybridization of the amplicons of the first encoded particle set with detectably labeled sample DNA and a second signal indicating specific hybridization of the amplicons of the first encoded particle set with detectably labeled reference DNA; and

comparing the first signal and the second signal to detect differences between the first and second signals, the differences of the first and second signals indicative of differences between the sample DNA and the reference DNA, thereby assaying the DNA sample.

2. The method of claim 1, wherein the amplicons have a length in the range of about 500-1200 nucleotides, inclusive.

3. The method of claim 1, wherein the detectably labeled sample DNA is detectably labeled DNA obtained from an individual subject.

4. The method of claim 3, wherein the detectably labeled sample DNA is detectably labeled genomic DNA obtained from an individual subject.

5. The method of claim 1, wherein the detectably labeled sample DNA is human DNA.

6. The method of claim 1, further comprising:

providing a second encoded particle set comprising encoded particles having attached amplicons, the amplicons comprising random nucleic acid sequences together representing substantially an entire second template DNA sequence;

hybridizing the amplicons of the second encoded particle set with detectably labeled sample DNA;

hybridizing the amplicons of the second encoded particle set with detectably labeled reference DNA;

detecting a first signal indicating specific hybridization of the amplicons of the second encoded particle set with detectably labeled sample DNA and a second signal indicating specific hybridization of the amplicons of the second encoded particle set with detectably labeled reference DNA; and

comparing the first signal indicating specific hybridization of the amplicons of the second encoded particle set and the second signal indicating specific hybridization of the amplicons of the second encoded particle set to detect differences between the first and second signals, the differences of the first and second signals indicative of differences between the sample DNA and the reference DNA.

7. The method of claim 6, wherein the first and second encoded particle sets are provided in a mixture and further comprising:

associating encoding of the first encoded particle set with the first signal indicating specific hybridization of the amplicons of the first encoded particle set with detectably labeled sample DNA and a second signal indicating specific hybridization of the amplicons of the first encoded particle set; and

associating encoding of the second encoded particle set with the first signal indicating specific hybridization of the amplicons of the second encoded particle set with detectably labeled sample DNA and the second signal indicating specific hybridization of the amplicons of the second encoded particle set.

8. A method of assaying sample DNA, comprising:

providing a multiplex reagent comprising a mixture of two or more encoded particle sets encoded such that each particle of each encoded particle set is detectably distinguishable from each particle of each other encoded particle set, the encoded particles having attached amplicons amplified from a template DNA sequence, each encoded particle set having attached amplicons amplified from a different template DNA sequence compared to each other encoded particle set,

hybridizing the attached amplicons with detectably labeled DNA;

hybridizing the attached amplicons with detectably labeled reference DNA;

detecting a first signal indicating specific hybridization of the amplicons with detectably labeled DNA;

detecting a second signal indicating specific hybridization of the amplicons with detectably labeled reference DNA;

identifying the encoded particles so as to associate particle encoding with the first signal;

identifying the encoded particles so as to associate particle encoding with the second signal; and

comparing the first signal and the second signal for each encoded particle set, wherein differences in the first and second signals are indicative of differences between the sample and reference DNA, thereby assaying DNA.

9. The method of claim 8, wherein the amplicons attached to each encoded particle set comprise random nucleic acid sequences which together represent substantially an entire template DNA sequence, wherein the entire template DNA sequence is larger than each individual attached amplicon

10. The method of claim 9, wherein the entire template DNA sequence has a length in the range of about 20-300 kilobases, inclusive and each individual attached amplicon comprising a DNA sequence identical to a portion of the template DNA sequence and having a length in the range of about 500-1200 nucleotides, inclusive.

11. The method of claim 8, wherein the detectably labeled sample DNA is detectably labeled DNA obtained From an individual subject.

12. The method of claim 11, wherein the detectably labeled sample DNA is detectably labeled genomic DNA obtained from an individual subject.

13. The method of claim 8, wherein the detectably labeled sample DNA is human DNA.

14. A method of preparing an encoded bead set for assaying DNA, comprising:

a) performing a first amplification reaction using a DNA template and first reaction oligonucleotide primers, each of the plurality of first reaction primers comprising a variable non-specific degenerate DNA sequence and a contiguous constant DNA sequence, to yield a first reaction product comprising first amplicons, wherein each individual first amplicon comprises a DNA sequence identical to a random portion of the DNA template and a DNA sequence identical to the constant DNA sequence of the first reaction primers;

b) performing a second amplification reaction using at least a portion of the first amplicons as template DNA and second reaction oligonucleotide primers, the second reaction oligonucleotide primers comprising the constant DNA sequence of the first reaction primers, to yield a second reaction product comprising second amplicons, wherein each individual second amplicon comprises a DNA sequence identical to a random portion of the DNA template and a DNA sequence identical to the constant DNA sequence of the first reaction primers;

c) binding the second amplicons to a first plurality of encoded particles, yielding an encoded particle set for assaying DNA.

15. The method of preparing a reagent for assaying DNA of claim 14 wherein the second reaction oligonucleotide primers farther comprise a functional group for reaction with an encoded particle.

16. The method of preparing a reagent for assaying DNA of claim 14, further comprising repeating a)-c) using a second DNA template and binding second amplicons obtained thereby to a second plurality of encoded particles detectably different than the first plurality of encoded particles, yielding a second encoded particle set for assaying DNA.

17. The method of preparing a reagent for assaying DNA of claim 16, further comprising mixing the first encoded particle set and the second encoded particle set.

18. Tie method of preparing a reagent for assaying DNA of claim 16, further comprising repeating a)-c) using a third or subsequent DNA template and binding the third or subsequent amplicons produced thereby to a third or subsequent plurality of encoded particles, each of the third or subsequent plurality of encoded particles detectably different than each other plurality of encoded particles, yielding a third or subsequent encoded particle set for assaying DNA.

19. The method of preparing a reagent for assaying DNA of claim 18, further comprising mixing the first encoded particle set, the second encoded particle set, the third encoded particle set and/or subsequent encoded particle set.

20. A reagent for assaying DNA, comprising:

a plurality of encoded particles having attached amplicons amplified from a template DNA sequence, each individual attached amplicon comprises a DNA sequence identical to a random portion of the template DNA sequence, wherein the amplicons together represent substantially the entire template DNA and wherein the nucleic acid sequence identical to a random portion of the template DNA sequence of each individual amplicon is shorter than the entire template DNA.

21. The reagent of claim 20, wherein the entire template DNA sequence has a length in the range of about 20-300 kilobases, inclusive and each individual attached amplicon comprises a DNA sequence identical to a random portion of the template DNA sequence having a length in the range of about 500-1200 nucleotides, inclusive.

22. A multiplex reagent for assaying DNA, comprising:

a mixture of two or more pluralities of particles encoded such that particles of each plurality of particles are detectably distinguishable from particles of each other plurality of particles, the encoded particles having attached amplicons amplified from a template DNA sequence, each plurality of encoded particles having attached amplicons amplified from a different template DNA sequence compared to each other plurality of encoded particles, each individual attached amplicon comprising a DNA sequence identical to a random portion of the template DNA sequence.

23. A kit for assaying DNA, comprising:

a mixture of two or more pluralities of particles encoded such that particles of each plurality of particles are detectably distinguishable from particles of each other plurality of particles, the encoded particles having attached amplicons amplified from a template DNA sequence, each plurality of encoded particles having attached amplicons amplified from a different template DNA sequence compared to each other plurality of encoded particles, each individual attached amplicon comprising a DNA sequence identical to a random portion of the template DNA sequence