Methods for screening for gene specific hybridization polymorphisms (GSHPs) and their use in genetic mapping and marker development

• US 20070048768A1
• Filed: 06/22/2006
• Published: 03/01/2007
• Est. Priority Date: 06/30/2005
• Status: Abandoned Application

• Current Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Original Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Inventors: Zhu, Tong, Salmeron, John
• Application Number:
  US11/472,789
• Publication Number:
  US 20070048768A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• 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 for identification of gene specific hybridization polymorphisms (GSHPs) and their use is presented. The method involves the steps of a) global screening for hybridization polymorphisms using microarray; b) enzyme mediated genome complexity reduction; c) enzyme mediated differential signal amplification and noise reduction; d) data extraction and GSHP identification; and e) use of GSHPs in high throughput screening.

Claims

1. A method for the screening of genomic nucleic acid material for gene specific hybridization polymorphisms, the method comprising:

a) global screening for hybridization polymorphisms using microarray;

b) enzyme mediated genome complexity reduction;

c) enzyme mediated differential signal amplification and noise reduction;

d) data extraction and GSHP identification; and

e) use of GSHPs in high throughput screening.

2. A method for the screening of genomic nucleic acid material for gene specific hybridization polymorphisms, the method comprising:

a. selecting oligonucleotide probes and designing a microarray comprising said probes for the detection of sequence variation;

b. translating sequence variations into variations of hybridizing targets;

c. labeling and hybridizing the targets;

d. detecting a hybridization signal; and

e. quantifying the hybridization signal and detecting polymorphisms.

3. A method for detection of gene specific hybridization polymorphisms in polynucleotide sequences of genomic DNA, the method comprising:

a. selecting short oligonucleotide sequences complementary to the genomic polynucleotide sequences, said short oligonucleotide sequences to be synthesized directly onto or synthesized and placed onto a microarray surface;

b. preparing genomic DNA from two genetic sources and subjecting said genomic DNA to site-specific restriction using one or more restriction enzymes to produce restriction fragment length polymorphisms (RFLPs);

c. selectively amplifying RFLPs of a selected size range to create amplified polymorphism targets;

d. fragmenting the amplified targets randomly into fragments of from about 50 to about 200 bases and end-labeling the fragments unselectively;

e. hybridizing the end-labeled fragments to the short oligonucleotide sequences on the microarray surface; and

f. quantifying the signals from the hybridization and detecting polymorphisms.

4. The method of claim 3 wherein the short oligonucleotides selected in step a. are from about 25 mers to about 30 mers.

5. The method of claim 3 wherein the fragments of the amplified targets of step d. are end-labeled using fluorescence-tagged nucleotides and a terminal transferase.

6. The method of claim 3 wherein the hybridization signals of step f. are captured by a device selected from the group consisting of a laser scanner and a CCD.

7. The method of claim 6 wherein the captured hybridization signals are quantified using a computational algorithm.

8. The method of claim 3 further comprising:

g. comparing signals from different genetic backgrounds or varieties for signal differences and determining the origins of the differential signals.

9. The method of claim 8 further comprising:

h. identifying the single nucleotide polymorphisms that cause the differential signals of step g.

10. The single nucleotide polymorphisms identified in the method of claim 9.

11. A genetic map developed using the information generated in the method of claim 3.

12. A genetic map developed using the information generated in the method of claim 8.

13. Molecular markers developed using the information generated in the method of claim 3.

14. Molecular markers developed using the information generated in the method of claim 8.

15. A quantitative trait locus identified and defined using the information generated in the method of claim 3.

16. The quantitative trait locus of claim 15 further characterized using the molecular markers of claim 13.

17. The quantitative trait locus of claim 15 further characterized using the molecular markers of claim 14.