LABORATORY 3 & 4: COLONY LIFTS AND SOUTHERN ANALYSIS
Hybridization Analysis: Detection of Specific DNA Sequences
Background. Restriction digestion, electrophoresis and staining allow us to cut DNA molecules
into reproducible fragments and to look at the sizes of those fragments. However, it does not
give us much information about the DNA base sequence within the fragments. Often it is
important to know whether a specific DNA base sequence is present in a sample, and where it is
located with respect to restriction sites. The technique most commonly used to address these
problems is hybridization analysis.
Hybridization analysis involves separating (denaturing) the strands of the DNA
molecules to be analyzed and mixing those strands with a labeled, single-stranded DNA or RNA
probe. If the probe's sequence is complementary to a DNA molecule, then hybridization (or
annealing) can occur under the appropriate conditions. The sample can be rinsed to remove
excess probe and tested for the presence of hybridized probe. Hybridized probe indicates the
presence of the DNA sequence of interest.
One such application is the screening of DNA libraries. A DNA library is a collection of
clones that, taken together, represent the entire genome of an organism. To find a plasmid
containing a specific DNA sequence (or gene of interest), the entire library is transformed into a
bacterial host. The transformants are plated onto agar to produce individual colonies, each
representing one fragment of the organism's DNA, "lifted" onto a membrane, and the DNA
denatured and probed with the sequence of interest. If colonies are identified, they can be
transferred to separate cultures, and plasmid DNA prepared for further analysis and use.
Another major application of hybridization analysis is the testing of the products of a restriction
digest to determine which fragments, if any, contain a certain DNA sequence, or to determine the
size of a fragment containing that sequence. The procedure most commonly used for these is
called "Southern blotting" (after the man who invented it) and is followed by hybridization
analysis.
A. Make the probe.
a. Add 1g of the template DNA and sterile, double distilled water to a final
volume of 16 l.
b. Denature the DNA by heating in a boiling water bath for 10 min and
quickly chilling in an ice/water bath.
c. Mix DIG-High prime (vial 1) thoroughly and add 4l to the denatured
DNA, mix and centrifuge briefly
d. Incubate 1 h or ON at 37oC (during this wait, prepare your target)
e. Stop the reaction by adding 2 l 0.2M EDTA (pH 8.0) or heating to 65oC
for 10 min
B. Preparation of the target
a. Colony lift –
i. Carefully lay the membrane onto the pre-cooled agar plate.
Beginning at one edge of the plate lay the membrane down
smoothly, avoiding bubbles.
ii. Mark the membrane and plate with a distinctive pattern
iii. Remove the membrane from the plate after 1 min. a second lift
may be performed if necessary. Seal the master plate with
parafilm and store at 4oC until needed.
iv. Place the membrane, colony-side up, onto filter paper saturated
with 0.5 M NaOH/1.5 M NaCl. Incubate 5 min.
v. Briefly blot the membrane on dry filter paper.
vi. Place the membrane, colony-side up, onto filter paper saturated
with 1.5 M NaCl/0.5 M Tris-HCl, pH 7.4. Incubate for 5 min.
vii. Briefly blot on dry filter paper.
viii. Place the membrane, colony-side up, onto filter paper saturated
with 2X SSC for 5 min.
ix. UV cross link DNA (or bake for 30 min at 80oC) to bind the DNA
to the membrane.
b. Gel –
i. Take apart the transfer setup.
ii. Mark the location of a slot in the upper right-hand corner with a
pencil.
iii. Rinse 5 min in 2X SSC.
iv. Blot with filter paper.
v. UV cross-link (or bake for 30 min at 80oC) to bind the DNA to the
membrane.
C. Hybridization
Hybridization analysis is a little like fishing (for DNA). If you know a little about
the DNA sequence you're interested in, locating it shouldn't be too difficult. Using that
sequence, you can design a probe (the "hook") and the nitrocellulose-bound DNA
provides the pond. The rest is just methodology and it is the same for a gel or colony lift.
Prehybridization and hybridization of the membrane
i.
ii.
iii.
iv.
v.
vi.
Preheat an appropriate volume (10ml/100 cm2 filter) of DIG Easy Hyb
to hybridization temperature (to be announced).
Place the nitrocellulose membrane in a sealable bag or plastic container
(tupperware). Add enough prehybridization solution (i) to fully cover
the membrane and prehybridize 30 min, shaking gently, at the
hybridization temperature.
Denature DIG-labeled probe by boiling for 5 min and rapidly cooling in
ice/water bath.
Add the denatured DIG-labeled probe (iii) to preheated DIG Easy Hyb
(i) and mix well (avoid bubbles, they increase background).
Pour off prehybridization solution (ii) and add probe/hybridization
mixture (v) to membrane
Incubate 4 h to overnight, at the hybridization temperature with gentle
agitation.
Protocol adapted from DIG High Prime DNA labeling and Detection Kit 1 (Cat. No. 1
745 832) Roche Scientific.
Day 2. Washes and detection
D. Stringency washes
Rinse the filter as follows:
i. 2 X 5 min in 2X SSC, 0.1% SDS at RT. with constant agitation
ii. 2 X 15 min in 0.5X SSC, 0.1% SDS (pre-warmed to wash temperature) at 65oC
E. Detection
i. Rinse the membrane briefly (1-5 min) in Washing Buffer
ii. Incubate for 30 min in 100 ml Blocking solution.
iii. Incubate for 30 min in 20 ml Antibody solution
iv. Wash 2 x 15 min in 100 Washing buffer
v. Equilibrate 2-5 min in 20 ml Detection buffer.
vi. Incubate the membrane in 10 ml freshly prepared color substrate solution
in the dark. Do not shake during color development. Monitor the color
development (may take up to 16 h).
vii. Stop the reaction, when the desired spot or band intensities are achieved,
by washing the membrane for 5 min with 50 ml sterile double distilled
water or TE buffer.
viii. Results can be documented by photocopying the wet membrane or by
photography.
F. Using this information, you could select colonies for sequencing or other uses.
Colonies are selected using a sterile toothpick or pipetman tip, then dropping the
toothpick into a 3 ml volume of liquid nutrient media with antibiotic selection and
incubation at 37oC overnight.
Protocol adapted from DIG High Prime DNA labeling and Detection Kit 1 (Cat. No. 1 745 832)
Roche Scientific.
Conclusions for Laboratory 7 & 8:
Did the probe recognize any DNA fragments (or colonies) from the gel?
If so, what does that mean?