Screening a Library

Plate out library on nutrient agar in petri dishes.

Up to 50,000 plaques or colonies per plate

Cloning a Specific Gene

Approach depends on what you have as starting materials and what you know about the gene you’re trying to clone.

Conserved sequence probe

Oligonucleotide probe

Chromosome walking (Positional cloning)

Tagging

Conserved sequence probe:Probe = clone of a gene that is similar to the one you’re trying

to clone. Ex: homologous gene from another species.

Adjust hybridization conditions to permit hybridization even though probe sequence is <100% complementary to clone’s.

Can also be used to clone genes from the same species that code for similar, but distinct proteins.

Oligonucleotide probe:If you know at least part of protein’s amino acid sequence, can deduce possible DNA coding sequences (“reverse translation”).

Synthesize oligonucleotides with all possible codons’ sequences

Only one of the oligonucleotides in the mixture will be able to hybridize to the clone’s DNA, but that’s sufficient to identify the clone.

Use fragment ‘a’ as probeto rescreen library

Select clones that hybridize to ‘a’ probe that did not hybridize to ‘A’ probe

Repeat these steps to isolate series of overlapping clones

Eventually identify gene by molecularly mapping mutations

Chromosome walking:If know the approximate location of gene and have a probe from the general vicinity, can isolate a series of overlapping clones

until the gene is reached.

Tagging:Isolate a mutant allele of a gene caused by insertion of an extra piece of DNA into the gene. Use this DNA as probe to screen library made from mutant.

Ex:Transposons are mobile pieces of DNA that can jump from one place in the genome to another.

Isolate mutant allele that is caused by insertion of a particular transposon.

Make genomic library from mutant’s DNA.

Screen library with transposon DNA probe.

Isolate clone of mutant allele, then use DNA fragment adjacent to transposon to screen wild type library.

Can also use restriction fragments from a genomic clone as probes to isolate cDNA clones of a gene that is included in that genomic region.

And, vice versa, can use a cDNA clone of a gene as a probe to screen a genomic library in order to isolate clones that contain the entire gene, not just the exon sequences.