Vectors for larger DNA fragments

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Vectors for larger DNA fragments
? vectors - Can insert fragments of DNA up
to 25 kb. - Can introduce into cells at a
very high efficiency BAC vectors (bacterial
artificial chromosomes) - Contain sequences from
the E. coli F plasmid present at one copy per
cell. - Can clone up to 200 kb per BAC clone.
YAC vectors (Yeast artificial chromosomes) -
Contains sequences required to replicate and
maintain chromosome in budding yeast (like ?, end
up as a linear molecules) - a yeast origin of
replication, a centromere, and a telomere at each
end. - Can clone gt2,000 kb (2 Mb).
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• The DNA into which a foreign piece of DNA is
  cloned is called a VECTOR
• There are several classes of vectors in use
• Plasmids Accept up to 10 kb foreign DNA
• Phage ? 5-20 kb fragments (its own genome is
  only 50 kb!) Commonly used in making genomic
  libraries. (very high efficiency of transfection)
• Cosmids 35-45 kb similar to plasmids (high
  efficiency for transformations)
• YACs (Yeast Artificial Chromosomes) 300-2000 kb!
  (essential for cloning very large fragments)

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• Cloning Vectors
• plasmids
• viruses
• bacteriophage
• lambda (?)
• filamentous
• (ssDNA)
• combination

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? as a Cloning Vector

• infectious ? can be assembled in vitro
• foreign DNA can be incorporated into the ? genome
  
• non-essential genes removed
• phage assembly can occur with 40-52 kb of DNA
  (wild-type ? ? 50kb)

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Library Construction in ?

• Prepare foreign DNA
• Prepare vector DNA
• Mix vector, foreign DNA and ligase
• In vitro packaging
• Infect host E. coli
• Screen plaques
• Plaque purification
• Subclone fragment into plasmid

• Vector DNA
• purchase pre-cut and dephosphorylated

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Library Construction in ?

• Prepare foreign DNA
• Prepare vector DNA
• Mix vector, foreign DNA and ligase
• In vitro packaging
• Infect host E. coli
• Screen plaques
• Plaque purification
• Subclone fragment into plasmid

COSLLLLLLLLG AATTCFFFFFFFG AATTCRRRRRRRRR

LLLLLLLLCTTAA GFFFFFFFCTTAA GRRRRRRRRRCOS
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• Library Construction
• Prepare foreign DNA
• Prepare vector DNA
• Ligation reaction
• In vitro packaging
• Infect host E. coli (plate on lawn)
• Screen plaques
• Plaque purification
• Subclone fragment into plasmid

Plaque clear zone on bacterial lawn cause by
lytic phage
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Plaque Lift
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• Library Construction
• Prepare foreign DNA
• Prepare vector DNA
• Ligation reaction
• In vitro packaging
• Infect host E. coli (plate on lawn)
• Screen plaques
• Plaque purification
• Subclone fragment into plasmid

• punch out plaque(s) with Pasteur pipette
• elute phage particles from agar
• re-plate and re-screen as needed

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Plaque hybridization
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• Library Construction
• Prepare foreign DNA
• Prepare vector DNA
• Ligation reaction
• In vitro packaging
• Infect host E. coli (plate on lawn)
• Screen plaques
• Plaque purification
• Subclone fragment into plasmid

• amplify cloned phage
• purify phage DNA
• excise insert
• ligate into plasmid

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To Screen Again or To Re-Make the Library?

• no guarantee that clone of interest is in library
• statistical estimates
• of independent inserts
• identical clones from previous successful screen

N ln(1-p)/ln(1-n) N of recombinants to
examine p probability of detecting clone n
average size of insert/genome size
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base pairs per haploid genome
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Cosmids

• cosmid vectors are plasmids with cos sequences
• cos sequence permits in vitro packaging
• infection produces colonies

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Yeast Artificial Chromosomes (YACS)

• replicates as linear chromosome in yeast
• can incorporate 100 kb - gt2 Mb of foreign DNA
• vector contains
• bacterial ori and ampr
• yeast centromere and ARS
• ciliate telomere
• yeast selectable marker

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Constructing a Genomic Library - extract genomic
DNA - cut with a restriction enzyme (want only
partial cutting) - mix with an excess of plasmid
cut with the same enzyme - ligate - transfer
(transform) into bacteria.
Pick a 4-cutter enzyme ie. Hae III ? AGCT Partial
Digestion
why would you only want partial cutting of the
DNA?
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Select out pieces of 20 kbases long, by
electrophoresis
Digested DNA (smear, consisting of fragments of
many different sizes)
MW ladder
40 kb ?
clone into vector
30 kb ?
Make recombinant DNA in appropriate vector
-cut out use DNA from this region
20 kb ?
10 kb ?
5 kb ?
- you can obtain a collection of clones of
different sequences that include the entire
genome of the organism
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The Central Dogma
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Making cDNA
the reverse transcriptase is not highly
processive, so you end up with some
incompletely-synthesized first-strand DNA
normally RNAseH is used in this step
the 3 end of any particular ssDNA may not form
such a hairpin
one normally uses a mixture of random primers in
this step
this step is not necessary if you use random
primers
end result cDNA where not all molecules are
full-length
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Preparation of cDNA

• 1) Isolate mRNA
• 2) Synthesize DNA-RNA hybrid
• reverse transcriptase
• oligo-dT primer
• random priming
• 3) Synthesize 2nd DNA strand
• 4) Add termini

? RNA dependent DNA polymerase
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Self-Priming

• 1) Isolate mRNA
• 2) Synthesize DNA-RNA hybrid
• 3) Synthesize 2nd DNA strand
• self-priming
• replacement synthesis
• primed synthesis
• 4) Add termini

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Replacement Synthesis
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Primed Synthesis

• Terminal Transferase (TdT)
• adds dNTP to 3-OH
• end label probes
• homopolymer tails

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• 1) Isolate mRNA
• 2) Synthesize DNA-RNA hybrid
• 3) Synthesize 2nd DNA strand
• 4) Add termini
• homopolymer tails
• poly-C on insert
• poly-G on vector
• include restriction site in primer(s)
• add linkers

3-(T)18GAGCTCTGATCA(GA)10-5 XhoI SpeI
? XhoI