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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 allows scientists to isolate genes based on their 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 a chromosome and construct a high-resolution molecular linkage map. Through this process, closely linked molecular markers are identified, helping to pinpoint the target gene’s location, clone it, and ultimately understand its function and the biochemical mechanisms involved in related diseases.
The positional cloning process typically involves six main steps:
1. **Identifying molecular markers linked to the target gene** – This is done using near-isogenic lines or bulked segregant analysis (BSA) to detect markers that are closely associated with the gene of interest.
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 molecular marker linked to the target gene is used as a probe to screen the library and identify positive clones.
3. **Building a contig for the target region** – Using the ends of positive clones as probes, chromosome walking is performed to obtain large DNA fragments that span the region flanking the target gene.
4. **Fine mapping of the target region** – By integrating existing genetic maps and identifying new molecular markers, the resolution of both the genetic and physical maps is improved, allowing for more precise localization.
5. **Precise mapping and chromosome landing** – Flanking markers and mixed sample mapping help narrow down the exact location of the gene. Chromosome landing is then used to isolate the clone containing the target gene using markers on either side as probes.
6. **Isolating and identifying the target gene** – Positive clones may contain multiple candidate genes. These are identified through methods like cDNA library screening, sub-capture, and direct cDNA selection. Further analysis, including co-segregation, expression patterns, and sequence homology, helps confirm the target gene. Functional complementation experiments are often the most definitive way to validate the gene's role.