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LECTURE
CONNECTIONS
19 | Molecular Genetic Analysis and
Biotechnology
© 2009 W. H. Freeman and Company
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.
b.
c.
d.
forward genetics
reverse genetics
both forward and reverse genetics
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