Novel method for carrying out accurate chromosome counting by applying saccharum centromere specific repetitive sequence

A repeat sequence, centromere technology, applied in the fields of bioinformatics and molecular cytogenetics, can solve problems such as difficulty in obtaining centromere sequences

Active Publication Date: 2021-07-06
NANTONG UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Recent studies have shown that centromere repeat sequences may be very different among different species, and even cl

Method used

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  • Novel method for carrying out accurate chromosome counting by applying saccharum centromere specific repetitive sequence

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Effect test

Embodiment 1

[0031] Example 1: Implementation of ChIP-Seq Technology

[0032] ChIP-Seq technology refers to the method of high-throughput sequencing of DNA obtained after chromatin immunoprecipitation ChIP.

[0033] The basic process of ChIP-Seq used in the present invention is:

[0034] 1. Grind fresh and young leaves with liquid nitrogen to extract cell nuclei;

[0035] 2. Extract the nucleus, use micrococcal ribozyme to enzymatically hydrolyze the nucleus, and randomly break the chromatin into fragments smaller than 300bp;

[0036] 3. Add the antibody of centromere-specific histone CENH3, and bind to the target protein-DNA complex; divide the nuclei after enzymatic hydrolysis into three parts, one part is the internal reference (input), and one part is added to the pre-immune serum ( Mock), another CENH3-specific antibody was added to immunoprecipitate the protein-DNA complex, and inverted overnight at 4°C;

[0037] 4. Add Protein A to bind the antibody-target protein-DNA complex and...

Embodiment 2

[0050] Embodiment 2: Primer design and PCR amplification

[0051] Use the primer design software Primer Premier 5.0 to design primers for the centromere sequence CENTS. The set conditions are: first, the full-length or partial sequence of the nucleic acid sequence; second, the length of the primer is 21bp±5bp; third, the GC content of the primer sequence is 40-60%; the fourth is to avoid the formation of stable dimer or hairpin structure between the primers; the fifth is that the primers cannot be mismatched at the non-target site of the template sequence.

[0052] The conditions of PCR amplification were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 45 s, annealing at 57°C for 30 s, extension at 72°C for 90 s, 30 cycles, and final extension at 72°C for 7 min.

[0053] It should be noted that the band amplified by PCR should be a single band. The PCR product can be directly purified and recovered using the QIAGEN kit, the product number: 28104, name: QIAquick ...

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Abstract

The invention relates to the technical field of bioinformatics and molecular cytogenetics, in particular to a novel method for carrying out accurate chromosome counting by applying a saccharum centromere specific repetitive sequence. The method comprises the following steps: step 1, acquiring the saccharum centromere specific repetitive sequence; and step 2, separating the saccharum centromere repetitive sequence and carrying out counting analysis. According to the invention, a method of combining chromatin immunoprecipitationassay with a high-throughput sequencing technology (ChIP-Seq) for a saccharum centromere specific histone is utilized to obtain a DNA sequence of a saccharum centromere region; and through comparative analysis, repetitive sequences in centromere regions of different saccharum species are screened out. Primers are designed according to the sequence, amplified and marked to be used for mitosis metaphase fluorescence in situ hybridization (FISH), and chromosome counting is carried out. Result shows that the sequence generates specific, clear and bright signals in centromere regions of all chromosomes of different saccharum species. The number of chromosomes of different materials can be rapidly and accurately obtained by counting the number of the centromere signals.

Description

technical field [0001] The invention relates to the technical fields of bioinformatics and molecular cytogenetics, in particular to a new method for accurate counting of chromosomes using sugarcane centromere-specific repeat sequences. Background technique [0002] Sugarcane (Saccharum spp.) is an important sugar crop and bioenergy crop in my country and even in the world. At present, it is generally accepted that Saccharum is mainly divided into six species: two wild species are cut hands dense species and large-stem wild species, and four cultivated species are tropical species, Chinese species, Indian species and fleshy spike species. Studies have confirmed that all Saccharum species are polyploid, with a large number of chromosomes and large variations. Among them, the number of chromosomes in the materials that have been found in the cut hand dense species ranges from 40 to 128, and there are about 40 genotypes, which is the most variable species in the genus Saccharum...

Claims

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Application Information

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IPC IPC(8): C12Q1/686
CPCC12Q1/686
Inventor 王凯张会汪保华
Owner NANTONG UNIVERSITY
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