Lambda and alternative vectors

Question 1

cDNA: generally short; –> plasmids (cloning large inserts)

Answer 1

• Tissue-specific mRNA libraries
• Size fractionated mRNA libraries
• Small RNA libraries
• Requires RT step

Question 2

Eukaryotic genomic DNA (gDNA): larger; x plasmids (cloning large inserts)

Answer 2

• Whole genome cloning
• gDNA genes, intergenic regions
• Repetitive regions X plasmids

Question 3

Phagemid

Answer 3

• 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

Question 4

Cosmid

Answer 4

• 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

Question 5

Fosmid

Answer 5

• uses fertility plasmid (F plasmid) which encodes pilus
• insert size: 30-44kb
• low copy number (1-2 per cell)

Question 6

PAC (P1-derived Artificial Chromosome)

Answer 6

• P1 phage-based plasmids: the phage replicates as a plasmid
• low copy number (1-2 per cell)
• insert size: 130-150kb

Question 7

BAC (Bacterial Artificial Chromosome)

Answer 7

• based on F plasmid ori
• low copy number (1-2 per cell)
• insert size: up to 300kb

Question 8

YAC (Yeast Artificial Chromosome)

Answer 8

• Saccharomyces sereviciae centromeres, telomeres and yeast ori (autonomous replicating sequence)
• low copy number
• insert size: 0.2-2.0Mb

Question 9

Bacteriophages

Answer 9

• 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

Question 10

T4 Phage Genome Expression

Answer 10

• 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

Question 11

Bacteriophage Lambda

Answer 11

• 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

Question 12

Lambda genome

Answer 12

• coat proteins
• non-essential region: integration and recombination (lysogeny)
• DNA synthesis, host cell lysis, and pathway regulation

Question 13

Lambda as a vector

Answer 13

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

Question 14

Lambda vectors

Answer 14

• 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

Question 15

Cosmid cloning

Answer 15

• 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

Question 16

Cosmids vs Plasmids & Phage

Answer 16

• Larger capacity (up to 44kb-moderate size)
• Choice of cloning pathway
• Choice of selection systems for recombinants
• Simple colony selection
• but repetitive DNA (euk) in high copy number cosmids poorly-tolerated in E. coli
• Often undergo deletions and rearrangements

Question 17

BACs & Fosmids

Answer 17

• F-plasmid of E. coli: parA & parB maintain low copy number
• Very high insert capacity (~300kb)
• Require electroporation
• Very stable
• F-plasmid genes in cosmids = fosmids
• Cosmid capacity (30-44 kb) with F plasmid stability

Question 18

Yeast Artificial Chromosomes (YACs)

Answer 18

• 1-2 molecules per cell.
• 1x centromere, 2x telomeres and 1x autonomous replicating sequence (ARS)
• <2 Mb inserts (but picky insert positioning)
• adenine mutant yeast spheroplasts: adenine deficiency (red/white selection), external cell wall removed, embedded in agar for stability
• Very low transformation efficiency & DNA yield (~1 per cell)