Lambda and alternative vectors Flashcards
1
Q
cDNA: generally short; –> plasmids (cloning large inserts)
A
- Tissue-specific mRNA libraries
- Size fractionated mRNA libraries
- Small RNA libraries
- Requires RT step
2
Q
Eukaryotic genomic DNA (gDNA): larger; x plasmids (cloning large inserts)
A
- Whole genome cloning
- gDNA genes, intergenic regions
- Repetitive regions X plasmids
3
Q
Phagemid
A
- a plasmid with f1 phage ori
- replicates as a plasmid
- packages as a ssDNA in e.g. M13
- often high copy number
- insert size: usually up to 5-10kb
4
Q
Cosmid
A
- a plasmid with a cohesive end (cos; ~200bp) sequence from l phage to allow l phage packaging
- can also have an ori (SV40, ColE1, f1)
- high copy number
- insert size: 30-44kb
5
Q
Fosmid
A
- uses fertility plasmid (F plasmid) which encodes pilus
- insert size: 30-44kb
- low copy number (1-2 per cell)
6
Q
PAC (P1-derived Artificial Chromosome)
A
- P1 phage-based plasmids: the phage replicates as a plasmid
- low copy number (1-2 per cell)
- insert size: 130-150kb
7
Q
BAC (Bacterial Artificial Chromosome)
A
- based on F plasmid ori
- low copy number (1-2 per cell)
- insert size: up to 300kb
8
Q
YAC (Yeast Artificial Chromosome)
A
- Saccharomyces sereviciae centromeres, telomeres and yeast ori (autonomous replicating sequence)
- low copy number
- insert size: 0.2-2.0Mb
9
Q
Bacteriophages
A
- RNA or DNA (ss or ds)
- 3.5 kb (MS2) – 280 kb
- Rely on host enzymes for replication
- Phage genes absent from host: enzymes (early), capsid proteins (late)
- Tailed phage: lytic life cycle
- Non-tailed phage: genome integrating (prophage), particles released on stress, non-lytic
10
Q
T4 Phage Genome Expression
A
- phage DNA replication: nucleases, DNApol and new sigma factors form phage DNA
- phage head proteins link with tail, collar, base plate, and tall fiber proteins
- self assembles; forms mature phage particle
- T4 lysozyme production at end of self assembly
11
Q
Bacteriophage Lambda
A
- Discovered by Esther Lederberg (1950)
- Infects E. coli via maltose transporter
- Temperate: lysogenic = prophage –> lytic
- 48.5kb genome: packages as a ssDNA, replicates as pDNA, switches to Rolling circle amplification, linearises at cos sites (200 nt length, 12 bp overhang), linear fragment inserted in the head
12
Q
Lambda genome
A
- coat proteins
- non-essential region: integration and recombination (lysogeny)
- DNA synthesis, host cell lysis, and pathway regulation
13
Q
Lambda as a vector
A
Central ~1/3 genome is non essential: ~23kb
- Replace by stuffer DNA
- Replace by DNA fragment (random)
- Replace by expression cassette
Distance between 2 cos sites must be between ~35kb and ~53kb
- Upper and lower packaging limits of lambda
- Efficiency dramatically reduced or lost
14
Q
Lambda vectors
A
- digestion causes fragment with restriction enzyme sites
- lambda arms attached at each side of stuffer fragment: <23kb insert
- non-recombinant -
too small for packaging - recombinant –
efficient packaging
15
Q
Cosmid cloning
A
- Hybrids of plasmids and lambda vectors: plasmids + cos site, in vitro l replication/packaging system, <40 kb inserts
- Concatemers at 35-53 kb cosmid size
- Cloning options: package phage in vitro and infect E. coli, transform E. coli with cosmid DNA
- Must propagate in E. coli: No lambda genes