Synthesis of long fish probes

• US 9,580,746 B2
• Filed: 01/02/2014
• Issued: 02/28/2017
• Est. Priority Date: 03/05/2013
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Leproust, Emily Marine, Chen, Siyuan, Ruvolo, Michael
• Primary Examiner(s): THOMAS, DAVID C
• Application Number:
  US14/146,216
• Publication Number:
  US 20140256575A1
• Time in Patent Office: 1,153 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/10 Gene or protein expression profiling; Expression-ratio estimation or normalisation

Abstract

A method comprising: synthesizing a set of overlapping oligonucleotides that comprises probe sequences that hybridize to unique sequences in a chromosome, assembling the overlapping oligonucleotides in a way that produces one or more double stranded polynucleotides that each comprises multiple probe sequences, labeling the one or more double stranded polynucleotides to produce one or more labeled probes, and hybridizing the labeled probes to an intact chromosome, in situ, is provided.

Claims

1. A method comprising:

(a) synthesizing a set of overlapping oligonucleotides that comprises probe sequences that hybridize to unique sequences in a chromosome;

(b) assembling the overlapping oligonucleotides in a way that produces one or more double stranded polynucleotides that each comprises multiple probe sequences, wherein the contiguous nucleotide sequence of each of said one or more double stranded polynucleotides is at least 95% identical to a sequence of said chromosome;

(c) labeling the one or more double stranded polynucleotides to produce one or more labeled probes; and

(d) hybridizing said labeled probes to an intact chromosome that comprises repeat sequences, in situ.

2. The method of claim 1, wherein said assembling is done by ligating multiple double stranded oligonucleotides to one another.

3. The method of claim 1, wherein said assembling is done by polymerase chain assembly.

4. The method of claim 1, wherein said labeling is done by random priming.

5. The method of claim 1, wherein said labeling is done by nick translation.

6. The method of claim 1, wherein said labeling is done by conjugating one or more labels to said one or more double stranded polynucleotides.

7. The method of claim 1, wherein said probe sequences are in the range of 10-150 nucleotides in length.

8. The method of claim 1, wherein said one or more double stranded polynucleotides are in the range of 300 to 5,000 base pairs in length.

10. The method of claim 1, wherein said chromosome is a mammalian chromosome.

11. The method of claim 1, further comprising:

(e) reading the product of step (d) using a microscope to produce a hybridization pattern.

12. The method of claim 11, further comprising:

(f) comparing said hybridization pattern to a control hybridization pattern.

13. The method of claim 12, wherein said comparing identifies a chromosomal rearrangement.

9. A method comprising:

analyzing a genomic sequence to identify target regions that are separated by repeat sequences;

designing a set of overlapping probe sequences for each of said target regions;

synthesizing oligonucleotides that comprise said overlapping probe sequences; and

assembling the overlapping probe sequences in a way that produces, for each of said target regions, a double stranded polynucleotide, wherein the contiguous nucleotide sequence of said double stranded polynucleotide for each of said target regions is at least 95% identical to the sequence of said target region.