-:POSITIONAL CLONING AND IT’S USES IN MAPPING:- Represented by Sumi Choudhury

Positional cloning is a method of gene identification in which a gene for a specific phenotype is identified only by its approximate chromosomal location (candidate region). Initially, the region can be defined using techniques such as linkage analysis. Positional cloning is then used to narrow the candidate region until the gene and its mutations are found.

DNA fragments from the closest known genetic marker are progressively cloned and sequenced A contig map of the locus is produced by chromosome walking. When the DNA clone is at or close to the mutant allele, the recombination frequency should be close to zero. If the chromosome walk proceeds through the mutant allele, the new polymorphisms will start to show increase in recombination frequency. Sequence comparison between wild type and mutant DNA in that region is then required to locate the DNA mutation that causes the phenotypic difference.

Reference : Abdelnoor, Ricardo & Yule, Ryan & Elo, Annakaisa & Christensen, Alan & Meyer-Gauen, Gilbert & Mackenzie, Sally. (2003). Abdelnoor RV, Yule R, Elo A, Christensen AC, Meyer-Gauen G, Mackenzie SA. Substoichiometric shifting in the plant mitochondrial genome is influenced by a gene homologous to MutS. Proc Natl Acad Sci USA 100: Proceedings of the National Academy of Sciences of the United States of America /pnas

In Map-based cloning of Arabidopsis, the genetic locus of ehy5 mutation was first mapped between markers ER and T20P8 on chromosome 2.Fine mapping was done using genetic markers designed from BAC clones. Sequence of the genomic DNA fragment from wild- type (WS) and ehy5 mutant plants and comparison with wild-type (Col) genomic DNA sequence indicate C to T mutation.

Reference : Prasad, Rajendra & V Kumar, Selva & Nandi, Ashis & Chattopadhyay, Sudip. (2012). Functional interconnections of HY1 with MYC2 and HY5 in Arabidopsis seedling development. BMC plant biology / The direction of the BAC clone is indicated by the arrow. The numbers above and below the arrow indicate the marker number and the corresponding recombinants for the respective marker.

It is a laboratory technique used to locate the position of a disease-associated gene along the chromosome. This approach works even when little or no information is available about the biochemical basis of the disease.

Positional cloning of Cystic Fibrosis Gene Northern blot analysis reveals only one of candidate genes is expressed in lungs and pancreas. Every CF patient has a mutated allele of the CFTR gene on both chromosome 7 homologs.

Proving CFTR is the right gene - Creation of CRFT gene “knockout” in mouse to examine phenotype without CRFT gene Targeted mutagenesis-Introduce mutant CFTR into mouse embryonic cells in culture Rare double recombinant events with homologous wild-type CFTR gene are selected. Mutant cell is introduced into normal mouse embryos where they incorporate into germ line-”Knockout” mouse created.

Positional cloning is one of the most important molecular approaches to genetic disorders. Since its introduction in research practice in the early 1980s, it has been widely utilised for defining the molecular origin of genetic diseases with Mendelian traits and still represents a necessary research step in this area. It constitutes the so-called “reverse genetics”, a term that indicates how genotype identification is followed by phenotype characterisation, in contrast to “forward genetics”, in which the study of phenotype leads to genotype identification Why Positional Cloning is Important

ACKNOWLEDGEMENT I would like to express my special thanks to - PRINCIPAL: Dr.Amit Chakravarty, whose inspiration motivated me to prepare this enriched and excellent presentation on Positional Cloning and Uses In Mapping. I am also thankful to my mentor Dr. ANIRBAN MITRA and & all my friends who had not only co-operated with me but also gave their important & valuable suggestions…….