Overview of Cloning

Traditional Procedure

1. Isolate DNA – genomic DNA prep

2. prepare probe to identify gene (cloned gene, cDNA, oligonucleotide, etc.)

3. Digest DNA with restriction endonucleases, prepare Southern blot

– hybridize blot with labelled probe – confirm sequence present

4. Prepare library of cloned DNA sequences (phage vectors)

– screen with probe to identify sequence of interest

5. Restriction map cloned DNA – find location of gene

6. Subclone gene into plasmid vector

7. Characterize subclone – sequencing, mutagenesis, expression

PCR (polymerase chain reaction)

If DNA sequences flanking gene known, isolate gene by amplifying it from crude DNA preps. Can be subcloned or sequenced directly.

Advantages – fast & (usually) easy

– large amounts DNA from small samples

Disadvantages – must know partial sequence ahead of time

Using genome sequence databases

Search Genbank for sequence of interest

request clone from research group that contributed sequence