BT 210

Lecture Outline- Week 7

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Chromosome Walking and Gene Amplification

With our lecture this week, we are going to turn out attention away from whole genomes, and back to the question of how to go about studying a particular gene of interest.

I. Starting Point

A. You know where your gene is on the genome

1) your gene has been tightly linked to a pair of markers

2) markers are physically located on either side of the gene of interest.

B. You have constructed a genomic library

1) what is a library?

2) how is it made?

II. Physical Isolation of the Gene: Chromosome Walking

A. General principle: successive rounds of hybridization

1) RFLP #1 sequence serves as your initial probe

2) complementary genomic DNA isolated and sequenced

3) sequence from 3' end of hybridizing genomic DNA fragment used

as next probe

4) continue until RFLP #2 sequence is found

B. Gene sequences within the "walk" region are identified using a variety of techniques

1) inter-species sequence comparisons

2) ORF scanning

3) sequence comparisons between "wild type" and mutants

III. Gene Amplification

A. Two techniques: PCR and Cloning

1) we already know a lot about PCR

a. review how it works

b. how this is used for gene amplification

2) we will concentrate on cloning today

B. Cloning as a method of gene amplification

1) advantages and disadvantages

2) three basic steps:

a. cutting and joining of DNA

-- vector selection

b. introduction of recombinant molecules into host cells

-- why bacteria are popular host cells

c. selection of cells that have taken up recombinant molecules