Primer-directed chromosome painting

• US 8,034,917 B2
• Filed: 08/28/2008
• Issued: 10/11/2011
• Est. Priority Date: 08/28/2008
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Yamada, N. Alice
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US12/200,675
• Publication Number:
  US 20100055681A1
• Time in Patent Office: 1,139 Days
• Field of Search: None
• US Class Current: 536/24.31
• CPC Class Codes: C12Q 1/6841 In situ hybridisation

Abstract

An oligonucleotide composition is provided. The subject composition comprises: a mixture of at least 10 of sets of oligonucleotides, wherein each of the sets of oligonucleotides comprises at least 100 different oligonucleotides of the following formula: X₁—V—X₂, wherein: X₁ and X₂ provide binding sites for a pair of PCR primers and V is a variable region that has a variable nucleotide sequence that is complementary to one or more discrete regions of a mammalian genome; the nucleotide sequences of X₁ and X₂ are the same for each oligonucleotide of a set and different for oligonucleotides of different sets; and the variable regions of each set are complementary to different discrete regions of said mammalian genome. Methods of using the composition and kits containing the composition are also provided.

Claims

1. An oligonucleotide composition comprising:

a mixture of at least 2 sets of oligonucleotides,

wherein each of said sets of oligonucleotides comprises at least 10 different oligonucleotides of the following formula;

X₁—

V—

X₂, wherein;

X_i and X₂ provide binding sites for a pair of PCR primers;

V is a variable region that has a variable nucleotide sequence that is complementary to one or more regions of a mammalian genome;

the nucleotide sequence of X₁ is the same for each oligonucleotide of a set and different for oligonucleotides of different sets;

the nucleotide sequence of X₂ is the same for each oligonucleotide of a set and different for oligonucleotides of different sets; and

the variable regions of each set are complementary to a plurality of different regions of said mammalian genome.

2. The oligonucleotide composition of claim 1, wherein said mixture comprises at least 10 sets of oligonucleotides.

3. The oligonucleotide composition of claim 1, wherein each set comprises at least 1000 different oligonucleotides.

4. The oligonucleotide composition of claim 1, wherein, within a given set of oligonucleotides, the variable regions are complementary non-overlapping regions of a single chromosome.

5. The oligonucleotide composition of claim 1, wherein, within a given set of oligonucleotides:

a) at least some of the variable regions are complementary to one or more discrete regions of a first chromosome of said mammalian genome and b) at least some of the variable regions are complementary to one of more discrete regions on a second, different, chromosome of said mammalian genome.

6. The oligonucleotide composition of claim 1, wherein said oligonucleotides are 100-200 nucleotides in length.

7. The oligonucleotide composition of claim 1, wherein said binding sites are 15-30 nucleotides in length.

8. The oligonucleotide composition of claim 1, wherein said variable region is 30-60 nucleotides in length.

9. The oligonucleotide composition of claim 1, wherein said oligonucleotides are in solution.

10. The oligonucleotide composition of claim 1, wherein said composition is made by fabricating an array of said oligonucleotides using in situ synthesis methods;

and cleaving oligonucleotides from said array.

11. A labeling method comprising:

a) contacting a composition comprising a mixture of at least 2 sets of oligonucleotides,

wherein each of said sets of oligonucleotides comprises at least 10 different oligonucleotides of the following formula;

X₁—

V—

X₂, wherein;

X₁ and X₂ provide binding sites for a pair of PCR primers;

V is a variable region that has a variable nucleotide sequence that is complementary to one or more regions of a mammalian genome;

the nucleotide sequence of X₁ is the same for each oligonucleotide of a set and different for oligonucleotides of different sets;

the nucleotide sequence of X₂ is the same for each oligonucleotide of a set and different for oligonucleotides of different sets; and

the variable regions of each set are complementary to a plurality of different regions of said mammalian genome;

with a first pair of PCR primers under PCR conditions to provide a first set of amplification products, wherein said amplification products comprise the variable regions of a first set of oligonucleotides; and

b) labeling said amplification products with a first label to provide first labeled amplification products.

12. The method of claim 11, further comprising contacting the composition with a second pair of PCR primers under PCR conditions to provide a second set of amplification products, wherein said amplification products comprise the variable regions of a second set of oligonucleotides;

and

labeling said amplification products with a second label to provide second labeled amplification products.

13. The method of claim 12, further comprising contacting said first and second amplification products with an intact chromosome under hybridization conditions to provide a contacted chromosome.

14. The method of claim 13, further comprising imaging said contacted chromosome to provide an oligonucleotide binding pattern.

15. The method of claim 14, further comprising comparing said oligonucleotide binding pattern to a reference binding pattern to identify a chromosomal rearrangement.

16. The method of claim 11, wherein said labeled amplification products emit a signal that identifies the amplified variable regions.

17. The method of claim 13, wherein said first labeled amplification products and said second labeled hybridization products hybridize to the same region of a chromosome to produce a composite signal that indicates the identity of said region.

18. A kit comprising

a) an oligonucleotide composition comprising a mixture of at least 2 sets of oligonucleotides,

wherein each of said sets of oligonucleotides comprises at least 10 different oligonucleotides of the following formula:

X₁—

V—

X₂, wherein;

X₁ and X₂ provide binding sites for pair of PCR primers;

V is a variable region that has a variable nucleotide sequence that is complementary to one or more regions of a mammalian genome;

the nucleotide sequence of X₁ is the same for each oligonucleotide of a set and different for oligonucleotides of different sets;

the nucleotide sequence of X₂ is the same for each oligonucleotide of a set and different for oligonucleotides of different sets; and

the variable regions of each set are complementary to a plurality of different regions of said mammalian genome; and

b) a plurality of pairs of PCR primers, wherein each pair of PCR primers amplifies the variable region from a different set of oligonucleotides to produce amplification products.

19. The kit of claim 18, further comprising labeling reagents for labeling said amplification products.