REAGENTS AND METHODS RELATING TO DNA ASSAYS USING AMPLICON PROBES ON ENCODED PARTICLES

• US 20130157899A1
• Filed: 02/21/2013
• Published: 06/20/2013
• Est. Priority Date: 12/05/2007
• Status: Active Grant

• Current Assignee: Revvity Health Sciences Incorporated (Revvity, Inc.)
• Original Assignee: PerkinElmer Health Sciences, Inc. (Revvity, Inc.)
• Inventors: Adler, Karl Edwin Jr., Schermer, Mack J.
• Granted Patent: US 9,127,307 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• 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 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.

2. The method of preparing a reagent for assaying DNA of claim 1, wherein the second reaction oligonucleotide primers further comprise a functional group for reaction with an encoded particle.

3. The method of preparing a reagent for assaying DNA of claim 1, 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.

4. The method of preparing a reagent for assaying DNA of claim 3, further comprising mixing the first encoded particle set and the second encoded particle set.

5. The method of preparing a reagent for assaying DNA of claim 3, 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.

6. The method of preparing a reagent for assaying DNA of claim 5, further comprising mixing the first encoded particle set, the second encoded particle set, the third encoded particle set and/or subsequent encoded particle set.

7. 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.

8. 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.

9. 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.

10. 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.