Transcript cDNA Libraries

Cosmids:
Plasmids that contain l cos sites.
This allows them to be packaged into l particles.
But don’t have l 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 Libraries
Collection 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 (l and cosmids) or use DNA to
transform E. coli (PACs and BACs).
Lambda Bacteriophage Vectors
cos
Selection:
cos
cos
Only recombinant phage produce plaques.
1. Size limits of DNA that fits into phage particle.
left l arm (15kb) + right l arm (10kb) +
insert (~15kb) = 40kb
2. Requirement for l genetic information:
cos sites & genes in right & left l 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
l: ~15 kb
Cosmids: ~35 kb
Human genome equivalents
~2 x 105 l clones
~8.6 x 104 cosmid clones
104 BAC clones (~300 kb/clone)
Recombinant DNA Libraries
Collection 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:
3’
Anneal oligo(dT) primers to
mRNAs.
Synthesize 1st cDNA strand
using Reverse Transcriptase.
1st cDNA strand
Degrade RNA with NaOH.
Synthesize 2nd cDNA strand
using DNA polymerase I.
Nick hairpin loop using S1
nuclease.
3.
Ligate with vector.
(plasmid or l)
2nd cDNA strand