Cosmids:Plasmids that contain cos sites.This allows them to be packaged into particles.But don’t have genes, therefore can clone larger insert fragments; 35-45 kb.

PACs:Based on P1 bacteriophage.Replicate as low copy number plasmids inside E. coli.Can clone inserts in 80-100 kb range.

BACs:“Bacteria Artificial Chromosomes”Based on 7 kb F1 plasmid (Ch. 7).Can clone inserts in 150-300 kb range.

Ways to get DNA into bacteria cells

Plasmids, BACs & sometimes PACs

Cosmids & sometimes PACs

Lambda

Recombinant DNAs

Recombinant DNA LibrariesCollection of many clones derived from a single DNA source.

Genomic Libraries:Many clones, each of which contains a fragment of chromosomal DNA from a particular species.

Complete genomic library: Entire genome is represented in at least one clone.

Making a Genomic Library

1. Digest chromosomal DNA & vector DNA with restriction enzyme(s).

2. Isolate desired size range of chromosomal DNA fragments.(Gel Electrophoresis)

3. Ligate chromosomal restriction fragments with vector DNA.

4. Either package clones ( and cosmids) or use DNA to transform E. coli (PACs and BACs).

Lambda Bacteriophage Vectors

cos cos

cosSelection:

Only recombinant phage produce plaques.1. Size limits of DNA that fits into phage particle.

left arm (15kb) + right arm (10kb) + insert (~15kb) = 40kb

2. Requirement for genetic information:cos sites & genes in right & left arms. Library

Complete Genomic Library

Entire genome is represented

Mininum number of clones (genome equivalent) needed depends on size of genome & vector used.

Example: Human genome = 3 x 106 kb

Average sizes of inserts

: ~15 kb Cosmids: ~35 kb

Human genome equivalents

~2 x 105 clones ~8.6 x 104 cosmid clones

104 BAC clones (~300 kb/clone)

Recombinant DNA LibrariesCollection of many clones derived from a single DNA source.

Genomic Libraries:Many clones, each of which contains a fragment of chromosomal DNA from a particular species.

Complete genomic library: Entire genome is represented in at least one clone.

cDNA Libraries:cDNAs = DNA copies of RNA molecules.

cDNA libraries: Each clone contains DNA copy of an individual mRNA.

Very useful for studying just the part of a gene that is present in mRNA.

Making a cDNA Library

1. Prepare poly(A)+ RNA from desired source.

2. Synthesize cDNAs:

Anneal oligo(dT) primers to mRNAs.

Synthesize 1st cDNA strand using Reverse Transcriptase.

Degrade RNA with NaOH.

Synthesize 2nd cDNA strand using DNA polymerase I.

Nick hairpin loop using S1 nuclease.

3. Ligate with vector. (plasmid or )

1st cDNA strand

2nd cDNA strand

3’