We are manufacturer and supplier of Frothing Pitcher,Frothing Milk Pitcher,Milk Frothing Pitcher,Milk Pitcher Frother,Stainless Steel Milk Jug. And we located in Jiangmen, Guangdong, China. If any interested, please contact us for free.
Frothing Pitcher,Frothing Milk Pitcher,Milk Frothing Pitcher,Milk Pitcher Frother,Stainless Steel Milk Jug Jiangmen Xinhui Zhancha Metal Products Co,. Ltd. , https://www.zchardware.com
Map clone of plant gene isolation
Positional cloning, also known as map-based cloning, was first introduced by Alan Coulson from the University of Cambridge in 1986. This technique involves identifying a gene based on its stable location within the genome. By analyzing genetic linkage or chromosomal abnormalities in a specific population, researchers can narrow down the gene's position on the chromosome and construct a high-resolution molecular linkage map. This process helps identify molecular markers closely associated with the target gene, further narrowing the candidate region for gene isolation and functional analysis, ultimately revealing its role in biological processes or disease mechanisms.
Positional cloning typically follows six key steps:
1. **Identifying molecular markers linked to the target gene**: Researchers use near-isogenic lines or bulked segregant analysis (BSA) to detect markers that are closely associated with the gene of interest. This helps locate the gene within a specific genomic region.
2. **Constructing and screening a genomic library**: Large-insert vectors such as cosmids, yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs) are commonly used. A genomic library is then screened using a marker linked to the target gene as a probe to isolate positive clones.
3. **Building a contig of the target region**: The ends of the positive clones are used as probes to perform chromosome walking, gradually extending the genomic coverage until a large fragment containing both flanking markers is obtained.
4. **Fine mapping the target region**: By integrating existing genetic maps and discovering new molecular markers, the resolution of both the genetic and physical maps is increased, allowing for more precise localization of the gene.
5. **Refining the gene’s position and chromosome landing**: Using flanking markers and mixed sample mapping, the exact location of the gene is pinpointed. Chromosome landing is then performed using these markers as probes to isolate the clone containing the target gene.
6. **Isolating and verifying the target gene**: Positive clones may contain multiple genes, so cDNA libraries, sub-capture techniques, and direct cDNA selection are used to identify potential candidates. These candidates are validated through co-segregation, expression patterns, sequence homology, and other analyses. The most definitive confirmation comes from functional complementation experiments, where the gene's function is tested by restoring the mutant phenotype.
This method has been instrumental in identifying genes responsible for various traits and diseases, providing valuable insights into their molecular mechanisms.