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Preparation method of multi-primer oligo probes

A multi-primer and probe technology, which is applied in the field of preparation of multi-primer oligo probes, can solve the problems of inability to disassemble the probe and limit the application of oligo-FISH technology, and achieve the effect of improving the application ability

Inactive Publication Date: 2019-09-06
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current method of preparing oligo probes is that each oligo sequence library is amplified with a single primer pair. The disadvantage is that each oligo sequence library can only be prepared into one probe, and the inability of the probe to be disassembled is greatly limited. More detailed application of oligo-FISH technology in plant molecular cytogenetics research

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  • Preparation method of multi-primer oligo probes

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Embodiment 1

[0018] Embodiment 1: the preparation method of multi-primer oligo probe

[0019] 1. Preliminary design of the oligo sequence: based on the sequenced Saccharum genus SES208 genome, first use the RepeatMasker software to filter out the repetitive sequence of the genome and design the remaining sequence as a 59bp oligo sequence, compare the sequence back to the genome to remove multiple Align the oligo sequences at the sites (homology > 75%), and use Primer5 to calculate the Tm value and hairpin Tm value of each remaining oligo sequence, and those with a difference > 10°C are retained.

[0020] 2. Add primers at both ends of the oligo sequence: take a 2mb interval on Chromosome 1-8 as an independent probe, and add the same primer sequence F1 to the 5' end of the oligo sequence of each independent probe, Add different primer sequences R1-R8 to the 3' ends of the oligo sequences of different probes, and the oligo sequences of 8 intervals form an oligo sequence library and send it t...

Embodiment 2

[0022] Example 2: Multi-primer oligo-FISH technology

[0023] The multi-primer oligo-FISH technology refers to the addition of different primer sequences R1-R8 to the 3' end of the oligo sequence of different probes when designing the oligo sequence for the preparation and differentiation of probes and then combined with fluorescence in situ hybridization technology. The method can split an oligo probe sequence library into 8 small probes to improve the application ability of FISH technology in more precise molecular cytogenetics research

[0024] The application steps of multi-primer oligo-FISH of the present invention are:

[0025] 1. Preparation of metaphase chromosomes: take the young root tips of SES208 and put them into 2mM 8-hydroxyquinoline solution for two hours to pretreat to enrich metaphase cells; then fix the root tips with ethanol: acetic acid (3:1) fixative solution, The root cap and elongation zone were removed, and the meristematic zone was hydrolyzed in a mi...

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Abstract

The invention discloses a preparation method of multi-primer oligo probes. According to the method, oligo sequences are designed by the aid of a bioinformatical path, independent probes are divided according to positions of the probes in genomes, same primer sequences F1 are added onto the 5' ends of the oligo sequences of the all probes, different primer sequences (R1-R8) are added onto the 3' ends of the oligo sequences of different probes, so that amplification and differentiation of the probes are implemented, and eight different single-chain oligo probes can be prepared once in an artificially synthesized manner. According to the method, an oligo sequence base can be divided into eight probes, and the method solves the problem that an existing oligo sequence base cannot be divided. According to the preparation method of the multi-primer oligo probes, the application ability of an FISH technique in research of more delicate plant molecular cell genetics is improved, and further development of the FISH technique can be facilitated.

Description

technical field [0001] The invention relates to the field of preparation and application of FISH technology probes, in particular to a preparation method of a multi-primer oligo probe. Background technique [0002] Fluorescence in situ hybridization (FISH) emerged in 1982, and after more than 30 years of development, it has become one of the most important methods in the study of plant molecular cytogenetics. The principle is to prepare known sequences as probes with markers, hybridize with nucleic acids in cells or tissues based on the principle of complementary base pairing, and use specific wavelengths of light to excite markers to achieve precise positioning of known sequences . At present, FISH technology is widely used in plant genomics and genomics research. However, DNA probes available for research applications in non-model plants are very limited and their development is difficult and labor-intensive. This has greatly hindered the application of FISH technology ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6811
CPCC12Q1/6811
Inventor 王凯孟状
Owner FUJIAN AGRI & FORESTRY UNIV
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