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Novel high-resolution quantitative multicolor fluorescent in-situ hybridization method and application thereof

A fluorescence in situ hybridization and fluorescein technology, which is applied in the field of new high-resolution multicolor fluorescence inspection, can solve the problem of not being able to observe the changes of multiple related genes in cells at the same time, and achieve the effect of improving signal-to-noise ratio and efficiency

Inactive Publication Date: 2013-05-22
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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AI Technical Summary

Problems solved by technology

[0003] Currently, single-color or two-color fluorescent in situ hybridization detection methods commonly used in the market have high sensitivity and specificity, but only 1-2 kinds of probes can be used to detect 1-2 kinds of gene abnormalities in one experiment, and they cannot be observed at the same time Changes of multiple related genes in the cell

Method used

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  • Novel high-resolution quantitative multicolor fluorescent in-situ hybridization method and application thereof
  • Novel high-resolution quantitative multicolor fluorescent in-situ hybridization method and application thereof
  • Novel high-resolution quantitative multicolor fluorescent in-situ hybridization method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1: the preparation of C-myc gene probe

[0033] 1. Acquisition of probe DNA

[0034] 1) Streak inoculation: Three BAC strains carrying the C-myc gene were respectively operated according to the following steps. First, the BAC strains were streak-inoculated on an agar plate containing antibiotics (chloramphenicol), and cultured at 37°C. Leave overnight (about 14 hours).

[0035] 2) Initial culture: Pick a monoclonal strain with good growth status the next day, inoculate it in 2ml of LB liquid medium containing antibiotics (chloramphenicol), put it in a shaker at 37°C, set the shaker speed to about 200rpm, and culture 6-8 hours.

[0036] 3) Overnight culture: transfer each 1ml of the bacterial solution obtained in step 2) to 200ml of LB liquid medium containing antibiotics (chloramphenicol), place it on a shaker at 37°C with a rotation speed of 200rpm, and cultivate overnight (about 14-16 hours).

[0037] 4) On the next day, a large amount of DNA was extract...

Embodiment 2

[0080] Example 2: Preparation of Green-C-myc (green), PF555-P16 (yellow), PF590-RB1 (red), HyPer5-CycD ((purple), PF415-P53 (blue) probe combinations

[0081] 1. Acquisition of probe DNA

[0082] 1) Streak inoculation: Streak inoculate BAC strains carrying C-myc, P16, RB1, CycD and P53 genes respectively on agar plates containing antibiotics (chloramphenicol), and culture overnight at 37°C (about 14 Hour).

[0083] 2) Initial culture: Pick a monoclonal strain with good growth status the next day, inoculate it in 2ml of LB liquid medium containing antibiotics (chloramphenicol), put it in a shaker at 37°C, set the shaker speed to about 200rpm, and culture 6-8 hours.

[0084] 3) Overnight culture: transfer each 1ml of the bacterial solution obtained in step 2) to 200ml of LB liquid medium containing antibiotics (chloramphenicol), place it on a shaker at 37°C with a rotation speed of 200rpm, and cultivate overnight (about 14-16 hours).

[0085] 4) On the next day, a large amoun...

Embodiment 3

[0127] Example 3: Detection of 20 genes on the same cell

[0128] 1. Acquisition of probe DNA

[0129] 1) Streak inoculation: Streak inoculate BAC strains carrying 20 genes (Table 3) respectively on agar plates containing antibiotics (chloramphenicol), and culture at 37° C. overnight (about 14 hours).

[0130] 2) Initial culture: Pick a monoclonal strain with good growth status the next day, inoculate it in 2ml of LB liquid medium containing antibiotics (chloramphenicol), put it in a shaker at 37°C, set the shaker speed to about 200rpm, and culture 6-8 hours.

[0131] 3) Overnight culture: transfer each 1ml of the bacterial solution obtained in step 2) to 200ml of LB liquid medium containing antibiotics (chloramphenicol), place it on a shaker at 37°C with a rotation speed of 200rpm, and cultivate overnight (about 14-16 hours).

[0132] 4) On the next day, a large amount of DNA was extracted and purified according to the operating instructions of the QIAGEN plasmid mass extra...

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Abstract

The invention relates to a novel high-resolution quantitative multicolor fluorescent in-situ hybridization method and application thereof. The method provided by the invention is implemented in a way that: a fluorescein label BAC (bacterial artificial chromosome) probe is adopted, and a continuous fluorescent in-situ hybridization method (probe elution rehybridization) is utilized; and after the hybridization each time, fluorescent information in six optical filter channels DAPI, Spectrum GreenTM, Cy3TM v1, Texas Red, Cy5 and PF-415 are acquired under a fluorescent microscope, and a computer automatic fluorescent visual field relocation method is utilized to accurately locate all the acquired images for all slices to the same visual field, thereby simultaneously analyzing 5-20 genes of a single normal or tumor cell. The method provided by the invention can screen and verify multiple gene mutants on the unicell level, and can be used for distinguishing different subtypes of multiple diseases as well as further classification of the same subtype of disease.

Description

technical field [0001] The invention relates to a novel high-resolution multicolor fluorescence inspection technology, in particular to establishing a quantitative multi-gene fluorescence in situ hybridization analysis method. The invention provides a method for screening and verifying multiple gene mutations at the single cell level, which can be used to distinguish different subtypes of multiple diseases and further classify the same subtype of diseases. Background technique [0002] Fluorescence In Situ Hybridization (FISH) technology is a molecular cytogenetics technology developed in recent years. Indirect labeling or known nucleic acid molecules directly labeled with fluorescein are used as probes. The probes and target sequence double-stranded DNA are denatured and then hybridized. Complementary heterologous single-stranded DNA molecules are annealed at a suitable temperature and ionic strength to form stable heterogeneous The source double-stranded DNA is collected ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 程涛安德士·莱特伯格胡林萍缪为民袁卫平
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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