The positoinal cloning, also known as map-based clonig, was first proposed by Alan Coulson of the University of Cambridge in 1986. The method of isolating genes according to the functional gene has a relatively stable locus in the genome, and based on the genetic linkage analysis of the isolated population or the chromosomal abnormality, the gene is clamped to a specific position of the chromosome, and a high density is constructed. The molecular linkage map finds the molecular markers closely linked to the target gene, narrows the candidate region and clones the gene, and clarifies its function and the biochemical mechanism of the disease.
The location cloning technology mainly includes the following six steps:
1. Screening for molecular markers linked to the target gene. The near-isogenic line of the target gene or the isolated population group analysis method (BSA) was used for linkage analysis to select the molecular markers of the local region where the target gene is located.
2. Build and screen a genomic library containing large inserts. Commonly used vectors include cosmid, yeast artificial chromosome (YAC), and P1, BAC, PAC and other bacterial-hosted vector systems. The genomic library was screened by using a molecular marker linked to the target gene as a probe to obtain a positive clone.
3. Construct a contig of the target gene region. The end of the positive clone was used as a probe genomic library, and chromosome walking was performed until a large fragment spanning group having molecular markers flanking the target gene was obtained.
4. Fine mapping of the region of the target gene. By integrating existing genetic maps and finding new molecular markers, the density of the dense spectrum of the genetic map and physical map of the target gene region is increased.
5. Fine mapping of target genes and chromosome landing. The flanking molecular marker analysis and mixed sample mapping were used to pinpoint the gene of interest. Then, the positive clone containing the target gene was obtained by chromosomal landing using the molecular markers on both sides of the target gene as probes.
6. Separation and identification of exons. Positive clones may contain multiple candidate genes. These candidate genes were identified by screening cDNA libraries, sub-capture and cDNA direct selection methods, and then co-segregation, spatiotemporal expression characteristics, homology comparison and other analysis to determine the target gene. Of course, the most straightforward proof is to perform functional complementation experiments.
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