How to pull out a specific genomic or cDNA from a library

There are many variations, here is one common scenario:

Working on a purified protein and want to find the gene or cDNA for it

-          purify protein and get some amino acid sequence from it

-          deduce which DNA sequence would produce this amino acid sequence

o       will be degenerate because several codons can code for one amino acid

-          construct degenerate primers

o       essentially a pool of all combinations of codons which could encode that amino acid sequence

o       can make short, single stranded nucleotide sequences on a machine

-          label the probe using with radioactivity or with fluorescent label

Hybridization

-          when two nucleic acid strands that were not originally together become basepaired

-          strands do not have to be identical to stably basepair

o       under “standard” conditions 15 contiguous bases pairing will form a stable duplex

o       however this varies according to

§         hybridization temp

§         salt concentration

o       can be DNA/DNA, DNA/RNA/ or RNA/RNA duplex

Library screening

-          your library is a bunch of plates with colonies

-          put nylon or nitrocellulose filter on top

-          some colonies stick to filter, some stay on plates

-          bust open phage, denature dsDNA, and bind to filter

-          incubate membrane in a solution containing the labeled probe under conditions where probe can hybridize to phage DNA on filter

o       some of the degenerate probe will be perfectly complementary to the DNA sequence of the actual gene

-          wash off unbound probe

-          Xray film on top, will be exposed where there is radioactivity

-          Black spot where phage containing gene is hybridized to the probe

-          Can do similar thing with plasmid-based libraries