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Molecular Diagnostics
Analysis and Characterization of
Nucleic Acids and Proteins
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Molecular Diagnostics
RESTRICTION NUCLEASE
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Different species of bacteria make different restriction
nucleases, which protect them from viruses by
degrading incoming viral DNA.
Each nuclease recognizes a specific sequence of four
to eight nucleotides in DNA.
These sequences, where they occur in the genome of
the bacterium itself, are protected from cleavage by
methylation at an A or a C residue
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Molecular Diagnostics
Type 1 restriction enzymes
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Have both nuclease and methylase activity
Complex enzymes with two subunits.
They bind to sites of 4–6 bp separated by 6–8 bp
and containing methylated adenines.
The site of cleavage can be over 1000 bp from
this binding site.
An example is EcoK from E. coli K 12.
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recognizes the site:
5 ‘- A C N N N N N N G T G C - 3’
3’ - T G N N N N N N C A C G – 5’
 and adenine residues (A) are methylated
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Type II restriction enzymes
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Used most frequently in the laboratory.
Do not have methylation activity
Bind as simple dimers to their symmetrical DNA
recognition sites (palindromic or bilateral
symmetry)
Cleave the DNA directly at their binding site,
producing fragments of predictable size.
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Type III restriction enzymes
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Able to both methylate and restrict (cut) DNA.
Complex enzymes with two subunits.
Recognition sites are asymmetrical
Cleavage occurs 24–26 bp from recognition site
to the 3 side.
Example is HinfIII from H. influenzae.
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It recognizes the site:
5’- C G A A T – 3’
3’- G C T T A – 5’
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adenine methylation occurs on only one strand.
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Restriction Endonucleases: Type II
Cohesive Ends
Cohesive Ends
Blunt Ends
(5´ Overhang)
(3´ Overhang)
(No Overhang)
BamH1
KpnI
HaeIII
GGATCC
CCTAGG
GGTACC
CCATGG
GGCC
CCGG
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Molecular Diagnostics
Restriction Endonucleases: Type II
GGATCC
CCTAGG
AGATCT
TCTAGA
BamHI
BglII
BssSI
TspRI
(5’ Overhang)
(5’ Overhang)
(5’ Overhang)
(3’ Overhang)
CTCGTG NNCAGTGNN
GAGCAG NNGTCACNN
Enzymes Generating
Compatible Cohesive Ends
Enzymes Recognizing
Non palindromic Sequences
CCCGGG CCCGGG
GGGCCC GGGCCC
GATC
CTAG
GGCC
CCGG
DpnI
HaeIII
SmaI
XmaI
(Requires methylation)
(Inhibited by methylation)
(Blunt Ends)
(5’ Overhang)
Methylation-sensitive Enzymes
Isoschizomers
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DNA ligase
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Catalyzes the formation of a phosphodiester
bond between adjacent 3-hydroxyl and 5phosphoryl nucleotide ends.
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Molecular Diagnostics
Restriction fragments can be religated
caccgtgGAATTCacgaacaa
gtggcacCTTAAGtgcttgtt
EcoRI
ligase + ATP
caccgtgG OH
gtggcacCTTAA
P
P
AATTCacgaacaa
HO Gtgcttgtt
ligase + ATP
caccgtgGAATTCtcgttgt
gtggcacCTTAAGagcaaca
acaacgaGAATTCctttatc
tgttgctCTTAAGgaaatag
EcoRI
acaacgaG OH
tgttgctCTTAA
ligase + ATP
P
P
AATTCctttatc
HO Ggaaatag
ligase + ATP
caccgtgGAATTCctttatc
gtggcacCTTAAGgaaatag
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Molecular Diagnostics
Self ligation can be prevented
caccgtgG OH
gtggcacCTTAA
P
P
AATTCacgaacaa
HO Gtgcttgtt
acaacgaG OH
tgttgctCTTAA
P
P
AATTCctttatc
HO Ggaaatag
phosphatase
caccgtgG OH
gtggcacCTTAA
AATTCacgaacaa
HO Gtgcttgtt
ligase + ATP
ligase + ATP
caccgtgG AATTCacgaacaa
gtggcacCTTAA Gtgcttgtt
Cannot be ligated
caccgtgGAATTCctttatc
gtggcacCTTAAGgaaatag
Nick
Cannot be ligated,
but can be replicated
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Molecular Diagnostics
The ends can be modified
caccgtgG
gtggcacCTTAA
AATTCacgaacaa
Gtgcttgtt
DNA pol
+ dNTPs
caccgtgGAATT
gtggcacCTTAA
AATTCacgaacaa
TTAAGtgcttgtt
ligase + ATP
caccgtgGAATTAATTCacgaacaa
gtggcacCTTAATTAAGtgcttgtt
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Molecular Diagnostics
Some enzymes have different recognition sites, but create
compatible cohesive ends
caccgtgGGATCCacgaacaa
gtggcacCCTAGGtgcttgtt
acaacgaAGATCTctttatc
tgttgctTCTAGAgaaatag
Bgl II
BamHI
caccgtgG OH
gtggcacCCTAG
P
P
GATCCacgaacaa
HO Gtgcttgtt
acaacgaA OH
tgttgctTCTAG
compatible cohesive ends
ligase + ATP
caccgtgGGATCTctttatc
gtggcacCCTAGAgaaatag
BamHI does not cut
BglII does not cut
P
P
GATCTctttatc
HO Agaaatag
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Molecular Diagnostics
Restriction Enzyme Mapping
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Digest DNA with a restriction enzyme.
Resolve the fragments by gel electrophoresis.
The number of bands indicates the number of
restriction sites.
The size of the bands indicates the distance
between restriction sites.
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Molecular Diagnostics
Restriction Enzyme Mapping
Two possible maps inferred from
the observations
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Restriction Enzyme Mapping
BamH1 XhoI
4.3 kb
3.7 kb
2.3 kb
1.9 kb
1.4 kb
1.3 kb
0.7 kb
BamH1
XhoI
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Homework?
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Determine the relative positions of HindIII and Sal I on
this piece of DNA
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Molecular Diagnostics
Hybridization Technologies
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Blots
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Southern blots
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Northern blots
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DNA immobilized on solid support
RNA immobilized on solid support
Western blots
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Proteins immobilized on solid support
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Southern Blot Hybridization
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Developed by Edwin Southern.
The Southern blot procedure allows analysis of
any specific gene or region without having to
clone it from a complex background.
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Transfer DNA from a gel matrix to a filter
(nitrocellulose, nylon)
Fix DNA to filter (Heat under a vacuum, UV cross-link
Block with excess DNA (unrelated)
Hybridize with labeled DNA probe
Wash unbound probe (controls stringency)
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Molecular Diagnostics
Blotting a Gel
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DNA Binding Solid Support
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Electrostatic and hydrophobic:
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Nitrocellulose
Nylon
Reinforced nitrocellulose
Electrostatic
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Nylon, nytran
Positively charged nylon
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Transfer of DNA to Membrane:
Capillary Transfer
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Transfer of DNA to Membrane:
Electrophoretic Transfer
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Transfer of DNA to Membrane:
Vacuum Transfer
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Molecular Diagnostics
Nucleic Acid Hybridization
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Takes advantage of the ability of individual singlestranded nucleic acid molecules to form doublestranded molecules (that is, to hybridize to each other).
Can occur between any two single-stranded nucleic
acid chains (DNA/DNA, RNA/RNA, or RNA/DNA).
The interacting single-stranded molecules must have a
sufficiently high degree of base complementarity.
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Molecular Diagnostics
Denaturation/Annealing: An Equilibrium
Reaction
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Molecular Diagnostics
Denaturation and Annealing of DNA
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Molecular Diagnostics
Melting Temperature (Tm)
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The temperature at which 50% of a nucleic acid is
hybridized to its complementary strand.
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Molecular Diagnostics
Melting Temperature and
Hybridization
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Your hybridization results are directly related to
the number of degrees below the melting
temperature (Tm) of DNA at which the
experiment is performed.
For an aqueous solution of DNA (no salt) the
formula for Tm is:
Tm = 69.3oC + 0.41(% GC)oC
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Molecular Diagnostics
Factors affecting melting temperature
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The energy required to separate two perfectly
complementary DNA strands is dependent on a number of
factors, notably:
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Strand length - long homoduplexes contain a large number of
hydrogen bonds and require more energy to separate them;
Base composition – sequences with high % GC composition are
more difficult to separate than those with a low % GC composition;
Chemical environment - the presence of monovalent cations
(e.g. Na+ ions) stabilizes the duplex, whereas chemical
denaturants (such as formamide and urea) destabilize the duplex
by chemically disrupting the hydrogen bonds.
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Molecular Diagnostics
Melting Temperatures: base
composition
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Sequences with high %
GC composition are more
difficult to separate than
those with a low % GC
composition
The following examples,
demonstrate the point.
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Tm = 69.3oC + 0.41(45)oC
= 87.5oC
Tm = 69.3oC + 0.41(40)oC
= 85.7oC
Tm = 69.3oC + 0.41(60)oC
= 93.9oC
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Molecular Diagnostics
Melting Temperatures: salts
composition
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Hybridizations though are always performed with salt.
the presence of monovalent cations (e.g. Na+ ions)
stabilizes the duplex, whereas chemical denaturants (such
as formamide and urea) destabilize the duplex by
chemically disrupting the hydrogen bonds.
The formula for the Effective Tm (Eff Tm).
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Eff Tm = 81.5 + 16.6(log M [Na+]) + 0.41(%G+C) - 0.72(%
formamide)
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Molecular Diagnostics
Melting temperature (Tm)
Hybrids
Tm (°C)
DNA-DNA
81.5 + 16.6 (log10[Na+]a) + 0.41 (%GCb) - 500/Lc
DNA-RNA or
RNA-RNA
79.8 + 18.5 (log10[Na+]a) + 0.58 (%GCb) + 11.8 (%GCb)2 - 820/Lc
oligod-DNA or
oligod-RNAd
For <20 nucleotides: 2 (ln)
For 20 35 nucleotides: 22 + 1.46 (ln )
• a Or for other monovalent cation, but only accurate in the 0.01-0.4 M range.
• b Only accurate for %GC in the 30% to 75% range.
• c L = length of duplex in base pairs.
• d Oligo, oligonucleotide; ln, effective length of primer = 2 × (no. of G + C) +
(no. of A + T).
• Note that for each 1% formamide, the Tm is reduced by about 0.6°C, while
the presence of 6 M urea reduces the Tm by about 30°C