Cotton somatic cell chromosome Oligo-FISH (oligonucleotide-fluorescence in situ hybridization) method

Abstract

The invention discloses a cotton somatic cell chromosome Oligo-FISH (oligonucleotide-fluorescence in situ hybridization) method. Compared with the prior art, the cotton somatic cell chromosome Oligo-FISH method is characterized in that according to the disclosed cotton genomic sequence information, a unpaired allosome oligonucleotide mixing pool is designed; the oligonucleotide mixing pool is subjected to emulsification PCR (polymerase chain reaction) amplification, in vitro transcription and inverse transcription to obtain a fluorescently-labeled ssDNA (single-stranded deoxyribonucleic acid) mixing pool, i.e., an oligonucleotide mixing probe of a target chromosome. The probe is used for performing FISH (fluorescence in situ hybridization) on the metaphase chromosome of the cotton somatic cell; the theoligo-FISH method is built; the technical support is provided for cotton chromosome DNA rearrangement, chromosome pairing, genetic relationship analysis, heterogenous chromatin identification and the like.

Description

technical field [0001] The invention belongs to the fields of bioinformatics and molecular cytogenetics, in particular to a cotton somatic cell chromosome Oligo-FISH method. Background technique [0002] Chromosome painting technology is one of the key technologies in the field of cytogenetics research, and it is an important means to accurately identify and distinguish chromosomes and their segments. Probe selection is a key link in the application of chromosome painting techniques. Typical painting probes come from cloned genome fragments or flow-sorted chromosomes, which are directly or indirectly fluorescently labeled by nick translation or PCR amplification. The length of the labeled probe is mostly 150-250bp, so that it is easy to penetrate into the fixed cells during the hybridization process. Since many genomic clones and flow-sorted chromosomes are rich in genomic repeats, the hybridization process usually requires blocking with untagged genomic repeats (Cot1DNA)....

AI Technical Summary

Problems solved by technology

Probes from a mixture of sequentially arranged clones on the chromosome can also be used to paint larger chromosomal regions, but this method is often challenging because each clone needs to be DNA extracted and labeled separately before hybridization. Due to random labeling and fragmentation, the stability of the efficiency is difficult to guarantee, and the application in complex plant genomes shows great limitations. Therefore, it is mostly used in plant species with relatively small genomes and low complexity.

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