Lambda and alternative vectors

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

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

• 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

• 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

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

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

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

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

• 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

• 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

• 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

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

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

• 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

• 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

• 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

• 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

• 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)