FCH 532 Lecture 5
New Homework assignment
Chapter 5
* In addition, only a fractional percentage of bases were
methylated (i.e. not every adenine was methylated, for
example) and these occurred at very specific sites in the
DNA.
* A characteristic feature of the sites of methylation, was
that they involved palindromic DNA sequences.
* In addition to possessing a particular methylase, individual
bacterial strains also contained accompanying specific
endonuclease activities.
* The endonucleases cleaved at or near the methylation
recognition site.
* These specific nucleases, however, would not cleave at
these specific palindromic sequences if the DNA was
methylated.
Thus, this combination of a specific methylase and
endonuclease functioned as a type of immune system for
individual bacterial strains, protecting them from infection by
foreign DNA (e.g. viruses).
* In the bacterial strain EcoR1, the sequence GAATTC
will be methylated at the internal adenine base (by the
EcoR1 methylase).
* The EcoR1 endonuclease within the same bacteria will
not cleave the methylated DNA.
* Foreign viral DNA, which is not methylated at the
sequence "GAATTC" will therefore be recognized as
"foreign" DNA and will be cleaved by the EcoR1
endonuclease.
* Cleavage of the viral DNA renders it non-functional.
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Figure 5-37 Restriction sites.
Types of restriction
endonucleases
• Type I and Type II restriction enzymes have
both endonuclease and methylase activity on
the same polypeptide.
• Type I enzymes cleave the DNA at a location of
at least 1000 bp away from the recognition
sequence.
• Type III enzymes cleave DNA from 24 to 26 bp
away from the recognition site.
• Type II restriction enzymes are separate from
their methylases. These enzymes cleave DNAs
at specific sites within the recognition sequence
(table 5-4).
Restriction endonucleases
recognize pallindromic sequences
• Palindrome-a word, verse, or sentence that reads the
same forwards or backwards.
• Restrction enzymes cleave 2 DNA strands at positions
that are symmetrically staggered about the center of the
palindromic sequence.
• Yields restction fragments with complementary singlestranded ends (1-4 nt in length called sticky ends).
• The sticky ends can associate by complementary base
pairing with other fragments generated by the same
restriction enzyme.
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Figure 5-37 Restriction sites.
Restriction maps
• After digest with DNA restriction endonuclease the fragments can
be separated according to size by gel electrophoresis.
• DNA can be separated according to size by agarose or
polyacrylamide.
• Duplex DNA is detected by staining with intercalating, planar,
aromatic cations such as ethidium, acridine orange, or
proflavin, between stacked base pairs. New stains like SYBR
are available that are notThese exhibit flurorescence under UV
light.
• As little as 50 ng of DNA may be detected in a gel by staining it
with ethidum bromide.
• Can also be used to visualize single stranded DNA or RNA.
• Can be used to generate a restriction map.
Figure 5-38 Agarose gel electrophoretogram of
restriction digests.
Digest of Agrobacterium
radiobacter plasmid pAgK84
digested with:
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A. BamHI, B. PstI, C. BglII, D.
HaeIII, E. HincII, F. SacI, G.
XbaI, H. HpaI. Lane I contains l
phage DNA digested with
HindIII as standards.
23130 bp
9416 bp
6557 bp
4361 bp
2322 bp
2027 bp
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Figure 5-39 Construction of a restriction map.
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Figure 5-40 Restriction map for the 5243-bp circular
DNA of SV40.
Restriction-fragment length
polymorphisms
• Individuality in species derives from genetic
polymorphism; homologous human chromosomes
differ in sequence ~every 1250 bp.
• Restriction enzyme digests of the corresponding
segments from homologous chromosomes contain
fragments of different lengths (restriction fragment
length polymorphisms or RFLPs) which can be used
for identification.
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Figure 5-41 Restriction-fragment length
polymorphisms.
Figure 5-42 Inheritance of RFLPs according to the
rules of Mendelian genetics.
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Circles females, squares males
Plasmid-based cloning vectors
• Plasmids - circular DNA duplexes of 1 to 200 kb that
contain the requisite genetic machinery (replication
origin) necessary for autonomous replication in bacteria
or yeast.
• Types of plasmids are determined by their copy number.
• Stringent control- one to a few copies per cell.
• Relaxed control- 10 to 700 copies per cell-if protein
synthesis is inhibited by an antibiotic (chloramphenicol),
the plasmid will continue to replicate up to 2000-3000
copies.
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Figure 5-43 The pUC18 cloning vector.
lac repressor
-galactosidase
permase
transacetylase
colorless
Plasmid-based cloning vectors
• Plasmids used in molecular cloning are
–
–
–
–
relatively small
replicate under relaxed control
carry genes for antibiotic resistance
number of restriction sites (polylinker) for inserting
DNA segments.
• Cannot be used to clone DNAs longer than ~10
kb.
• Blue-white screeing.
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Figure 5-46 Construction of a recombinant DNA
molecule.
Gene manipulation
• Restriction fragments with matching restriction sites are
annealed together and can be connected by the action of
ligase.
• If the foreign DNA and cloning vector have no common
restriction sites, they may still be spliced using a
terminal deoxynucleaotidyl transferase (terminal
transferase).
• Mammalian enzyme that adds nucleotides to the 3’-OH
group of a DNA chain. It is the only known DNA
polymerase that does not require a template.
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Figure 5-47 Splicing DNA using terminal transferase.
Gene manipulation
• Chemically synthesized pallindromic linkers may
also be used “insert” a required restriction site
onto a DNA sequence.
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Figure 5-48 Construction of a recombinant DNA
molecule through the use of synthetic oligonucleotide
adaptors.
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Figure 5-50 Detection of DNAs containing specific
base sequences by the Southern transfer technique.
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Figure 5-51 A degenerate oligonucleotide probe.
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Figure 5-52 Colony (in situ) hybridization.
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Figure 5-53 Chromosome walking.
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Figure 5-56
Constru
ction of a
recombinant
DNA molecule
by directional
cloning.
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Figure 5-54 The
polymerase chain
reaction (PCR).
•Thought up by Kerry Mullis
in 1985.
•Amplify DNA up to 10 kb.
•Heat denatured DNA is
incubated with
•DNA polymerase
•dNTPs
•Two oligonucleotide
primers
•Heat stable poymerases
used
•Taq
•Pfu
PCR
• Amplified DNA can be used for RFLP analysis, Southern
blotting, and sequencing.
• Can be used for rapid detection of diseases nad
mutations.
• Can be used to identify DNA from hair, sperm, blood by
amplification of short tandem repeats (STRs)-segments
of repeating DNA sequence (2 -7 bp) such as (CA)n and
(ATGC)n
• STRs are genetically variable and can be used as
markers for individuality. The number of tandem repeats
of STR are unique to an individual.
• STRs are amplified from unique sequence outside the
tandem repeats.
• RNA can be amplified by PCR; first reverse transcribing
it to DNA (cDNA) through reverse transcriptase.
Figure 5-57 Site-directed mutagenesis.
Allows for the “customization” of
a protein.
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Oligonucleotide containing a
short gene segment with the
desired altered base sequence
corresponding to the new amino
acid sequence is used as a
primer in the reaction.
In this case used DNA
polymerase I. Can also use
PCR to amplify a gene of
interest and insert a mutation in
the primer.
Production of proteins
• Cloned structural genes can be inserted into an
expression vector to produce recombinant protein.
• Relaxed control plasmid with an efficient promoter
can produce up to 30% of the total cellular protein
as the inserted structural gene.
• Inclusion bodies-large amounts of insoluble and
denatured protein. The protein must be extracted
and renatured by dissolving in a chaotrope like urea
or guanididium chloride and slowly renaturing the
protein.
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Figure 5-55 Electron micrograph of an inclusion body
of the protein prochymosin in an E. coli cell.
Production of proteins
• Can engineer a signal sequence to target the
protein to the periplasmic space of the bacteria so it
folds properly.
• Toxic proteins can be placed under an inducible
promoter (lac) promoter in a plasmid that also has
the gene for the lac repressor protein.
– Binding of the lac repressor will prevent the expression
from the lac promoter.
– After cells have grown to high density, an inducer
(isopropylthiogalactoside-IPTG, a synthetic
nonmetabolizable analog of allolactose) is added to
release the lac repressor protein.
Reporter genes can be used to
monitor transcription
• Rate at which a gene is expressed dependent on
upstream control sequences.
• Replace the gene you want to monitor with a reporter
gene.
• Reporter genes encode proteins that can be easily
detected by some assay. lacZ can be assayed with xgal and the production of blue color.
• Another reporter is the green fluorescent protein (GFP)
which produces a bioluminiscent protein when irradiated
with UV or 400nm light.
geneX
Replace geneX with reporter gene in the correct reading
frame
lacZ
In the presence of X-gal, expression will
produce the blue color.
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Figure 5-58 Use of green fluorescent protein (GFP) as
a reporter gene.
Transgenic organisms
• Organisms expressing a foreign gene are considered
transgenic.
• Foreign gene referred to as transgene.
• For the change to be permanent, transgene must be
stably integrated into germ cell.
• Established in mice by microinjection of DNA into a
pronucleus of a fertilized ovum.
• Can also be accomplished in an embryonic stem cell.
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Figure 5-59 Microinjection of DNA into the pronucleus
of a fertilized mouse ovum.
Nucleic acid sequencing
• Development of DNA sequencing techniques has
spurred the huge amount of DNA sequence data (>35
billion nucleotides in 2003 and growing!)
• Complete genomes determined for over 110 prokaryotes
and over 11 eukaryotes.
Nucleic acid sequencing
• Development of DNA sequencing techniques has
spurred the huge amount of DNA sequence data (>35
billion nucleotides in 2003 and growing!)
• Complete genomes determined for over 110 prokaryotes
and over 11 eukaryotes.