LECTURE CONNECTIONS 19 | Molecular Genetic Analysis and © 2009 W. H. Freeman and Company Biotechnology

19.3 Molecular Techniques Can Be Used to Find Genes of Interest - Shotgun cloning: one first clones a large number of DNA fragments, knowing that one or more contains the DNA of interest. - Gene library: a collection of clones containing all the DNA fragments from one source Creating a genomic DNA library

- cDNA library: consisting only of those DNA sequences that are transcribed into mRNA Creating a cDNA library Advantages: fragments from actively transcribed genes; and introns do not interrupt the cloned sequence. Disadvantages: contain only sequences that are presence in mature mRNA; and sequences expressed in the tissue from which RNA was isolated.

Gene Libraries Screening DNA libraries Plating clones of the library Probing plated colonies or plaques

In Situ Hybridization DNA probes used to determine the chromosomal location and to visualize a gene while it is in a cell. A labeled probe is applied to the slide. FISH (fluorescence in situ hybridization) is used to identify the chromosomal location of human genes. Determine the tissue distribution of specific mRNA molecules.

A) Deletion on one copy of chromosome 7 in humans. B) Presence of mRNA from the tailless gene in Drosophila.

Positional Cloning Isolating genes on the basis of their position on a genetic map. In Silico Gene Discovery: high-speed computerized DNA data mining. Application: isolating the gene for cystic fibrosis by positional cloning.

DNA Fingerprinting (DNA Profiling) DNA fingerprinting: the use of DNA sequences to identify individual persons. Microsatellites: variable number of copies of repeat sequences possessed by many organisms, which can be amplified by PCR. Application: identifying people who died in the collapse of the World Trade Center; identifying criminals; paternity tests; identify specific strains of pathogenic bacteria; etc.

19.5 Molecular Techniques Are Increasingly Used to Analyze Gene Function Forward genetics: –Begins with a phenotype to a gene that encodes the phenotype Reverse genetics: –Begins with a gene of unknown function, first inducing mutations and then checking the effect of the mutation on the phenotype

19.5 Molecular Techniques Are Increasingly Used to Analyze Gene Function Site-directed mutagenesis Reverse genetics: creating mutation in particular DNA sequences, and then studying the effects of these mutation on the organisms. Transgenic animals: animals that have been permanently altered by the addition of a DNA sequence to their genome.

19.5 Molecular Techniques Are Increasingly Used to Analyze Gene Function Knock out mice: a normal gene of the mouse has been fully disabled. Knock in mice: a mouse carries an inserted DNA sequence at specific locations.

19.5 Molecular Techniques Are Increasingly Used to Analyze Gene Function Silencing genes with RNAi Using RNAi for the treatment of human disease: lowering ApoB with RNAi Model Genetic Organism The mouse, Mus musculus

Concept Check 1 A geneticist is interested in the immune function of mice, and induces random mutations in a number of genes in mice and then determines which of the resulting mutant mice have impaired immune function. This is an example of ________. a.forward genetics b.reverse genetics c.both forward and reverse genetics d.neither forward nor reverse genetics

19.6 Biotechnology Harnesses the Power of Molecular Genetics Pharmaceuticals Human insulin Specialized bacteria Agricultural products Oligo nucleotide drugs Genetic testing Gene therapy Direct transfer of genes into humans to treat disease