bacteriophage RNA polymerases : promoters

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bacteriophage RNA polymerases promoters
T7 RNA polymerase TAATACGACTCACTATAGGGAGA
T3 RNA polymerase AATTAACCCTCACTAAAGGGAGA
SP6 RNA polymerase ATTTAGGKGACACTATAGAA
G not recognized by E. coli cellular RNA
polymerase active in vitro 30-40 mg RNA from 1
mg template DNA
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Ligation mechanism R ribose A adenine E
enzyme (ligase)
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The use of linkers short palindromic,
synthetic DNA sequences (self-complementary)

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The use of an adaptor presence/absence of the
5' phosphate group with respect to
ligation (no self-ligation of the sticky
ends)

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DNA cloning in a plasmid vector

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Use of T4 polymerase to couple DNA fragment (" L
I C ligation-independent cloning")
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Method of Henikoff to make systematic,
progressive deletions.
Controlled degradation by exonuclease III Remove
the second strand by nuclease S1 Circularize by
self-ligation with T4 DNA-ligase.
see also later - characteristics of
restriction enzymes - 'multiple cloning sites'
in vectors
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RESTRICTION ENDONUCLEASES Werner Arber,
Hamilton Smith, Daniel Nathans Nobel prize 1978

EOF efficiency of plating
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Infection efficiency of bacteriophage l on E.
coli strains (simplified description)
Infection of E. coli K E. coli B E. coli
C ------------------------------------------------
--------------------------------------------------
-------------------------- Phage from
E. coli K (lK) 1 10-3 1 E.coli B
(lB) 10-3 1 1 E. coli C
(lC) 10-3 10-3 1
Analysis of the relative "plaque forming units"
of phage l on different E. coli strains
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Characteristics of the different types (classes)
of restriction endonucleases

R.J. Roberts (CSHL) 10000 strains, 3000
restriction enzymes, 200 different recognition
sequences
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Class-IIs
Intron- and inteïn-encoded endonucleases
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Special endonucleases such as BcgI, AloI, BaeI,
PpiI, PsrI) excise small fragments of defined
length. Occurrence (pseudo-statistically)
once in 4 kb (if hexameric), once in 16 kb (if
heptameric). Example BcgI fragments are 32
nt long (after removal of the 3extensions).
Hence, 26 positions are N i.e. 426 possible
sequences (more than 4,5 x 1015). This allows
unique characterisation of a genome.
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Differences in kinetics not all target
positions are equally fast cleaved with some
enzymes, there is a high preference for some
sites, so that their use is hindered or limited
or unreliable. Star activity changing
buffer conditions (e.g. polarity) can result in
secondary cleavages. Sometimes
exploited to generate partial cleavages. .
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Bacterial genomes CCG and CGG are the rarest
trinucleotides in most AT rich bacterial
genomes. Hence, the restriction recognition
sequences contain these trinucleotides are
underrepresented. CTAG is the rarest
tetranucleotide in most GC rich bacterial
genomes. Examples of average fragment sizes
for the 3 categories of GC content in in
bacterial genomes are shown in this table.
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Tinkering at cleavage sites

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Cloning of PCR fragments engineering of
protruding ends

A restriction enzyme does not cleave at the
extreme end some extra base pairs are required
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Some examples of restriction sites, influenced by
methylation at dam-, dcm- or CpG-sequences.

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Quality control

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Specific 3-extensions by HphI
Specific 5-extensions by Tth111I Selective
radioactive labeling or chemical tagging (e.g.
by biotin).
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