Screening of diseased gene.pptx

Screening of diseased gene Lecture by: Dr. Navami Pai Positional cloning-Introduction Positional cloning is a laboratory approach used to locate the position of a disease-associated gene on a chromosome. Such a strategy can succeed even when nothing is known about...

Screening of diseased gene Lecture by: Dr. Navami Pai Positional cloning-Introduction Positional cloning is a laboratory approach used to locate the position of a disease-associated gene on a chromosome. Such a strategy can succeed even when nothing is known about the role of the gene’s encoded protein in the disease. The technique typically relies on the use of known polymorphic markers whose inheritance can be traced through various members of families affected by the disease. Steps involved in positional cloning It consists of different consecutive steps. A genetic analysis of the enrolled patients and their families is performed, to define a critical DNA region of interest. This analysis culminates in a statistical estimate of the probability that disease features may segregate in the families independently or in association with specific molecular markers located in known regions. In this latter case, a marker can be defined as being linked to the disease manifestations. RFLP Restriction fragment length polymorphism (RFLP) is a technique invented in 1984 by the English scientist Alec Jeffreys during research into hereditary diseases. It is used for the analysis of unique patterns in DNA fragments in order to genetically differentiate between organisms – these patterns are called Variable Number of Tandem Repeats (VNTRs). Genetic polymorphism is defined as the inherited genetic differences among individuals in over 1% of normal population. The RFLP technique exploits these differences in DNA sequences to recognize and study both intraspecies and  Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and highly locus-specific. An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus. Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP probes. The RFLP probes are frequently used in genome mapping and in variation analysis (genotyping, forensics, paternity tests, hereditary disease diagnostics) How it works  Eg: Detection of Sickle cell anemia by RFLP A mutation associated with a genetic disease may cause the loss or addition of a restriction site either within the gene or in a flanking region. A small number of RFLPs are associated with genes known to cause diseases, as the following example about sickle-cell anemia illustrates. In sickle-cell anemia (SCA), a single base-pair change in the gene for the β–globin polypeptide of hemoglobin results in an abnormal form of hemoglobin, Hb-S, instead of the normal Hb-A. Hb-S molecules associate abnormally, leading to sickling  The sickle-cell mutation changes an A–T base pair to a T–A base pair so that the sixth codon for β-globin is changed from GAG to GUG. As a result of this SNP allele, valine is inserted into the polypeptide instead of glutamic acid. THANK YOU