LECTURE CONNECTIONS

19 | Molecular Genetic Analysis and

© 2009 W. H. Freeman and CompanyBiotechnology

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