Lambda and alternative vectors Flashcards

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1
Q

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

A
  • Tissue-specific mRNA libraries
  • Size fractionated mRNA libraries
  • Small RNA libraries
  • Requires RT step
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2
Q

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

A
  • Whole genome cloning
  • gDNA genes, intergenic regions
  • Repetitive regions X plasmids
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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
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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
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5
Q

Fosmid

A
  • uses fertility plasmid (F plasmid) which encodes pilus
  • insert size: 30-44kb
  • low copy number (1-2 per cell)
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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
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7
Q

BAC (Bacterial Artificial Chromosome)

A
  • based on F plasmid ori
  • low copy number (1-2 per cell)
  • insert size: up to 300kb
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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
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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
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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
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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
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12
Q

Lambda genome

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

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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
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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
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16
Q

Cosmids vs Plasmids & Phage

A
  • 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
17
Q

BACs & Fosmids

A
  • 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
18
Q

Yeast Artificial Chromosomes (YACs)

A
  • 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)