Genetic Libraries

Part 1: How to build them

WHY DO WE WANT ONE?

WHAT GOOD IS IT?

IS IT WORTH THE EFFORT?

Suppose we want to study a

particular gene or protein from an

organism.

For example beta globulin

We will need lots of mRNA and protein

How do we get sufficient material for our experiments?

PCR works for DNA, not RNA.

1st step:Build a genetic library of that organism

Types

of

libraries

Genomic

cDNA

One type of

library is a

Genomic library

1. Get genome – DNA

Add some Restriction Enzymes to the mix

These will cleave the DNA into small pieces

All the fragments differ in size

So we can separate them with Gel Electrophoresis

Then we need to amplify them

Add DNA to plasmid

Insert into bacteria

Then you have a DNA library in many pieces

But when combined, contain the complete genome

Another

type of

library is a

cDNA

library

Just looks at expressed genes

A cDNA library is Tissue specific

Start with cell of interest (COI)

Start by isolating mRNA.

Use reverse transcriptase to make cDNA

Add restriction ends but don’t cut cDNA

Next we’ll need a

vector

--

lambda phage

Lambda phage sequence

Add cohesive ends to DNA segments

And insert into phage

Each phage containing DNA is allowed to infect an E. coli.

Get many copies when lise.

Lambda phages live a long time so good storage.

Genetic Libraries

Part 2: How to use them(coming soon)

CLONE

LIBRARY

SCREENING

FIRST SCREEN

USING

PLAQUES

Prepare a petri dish with an E. coli lawn

infect with phage

Phage plaques form on the lawn

Make radioactive probe with complimentary DNA probe

Isolate individual plaque (cDNA)

Use re to cut up cDNA small enough to fit in vector but not so small that genes get

too cut up.

may use two diif er to have cut in diff places.

insert into plasmid

add plamid to bacteria

end up w thousands of bac tea w diff sect of genome.

Southern and Northern Hybridization Analysis

Autoradiography