Southern Transfer and Hybridization

Labeling Probe and Southern Hybridization

Background:
The process of labeling and hybridization of Southern blots is performed in a series of
steps over several days. The first step is called prehybridization. During this step the
membrane containing the DNA is pretreated with a buffer containing blocking reagents
such as albumin or salmon sperm DNA. These treatments block any nonspecific hot
spots on the membrane that might bind to probe nonspecifically. !ften" prehybridization
is performed for #$ min using the same buffer that will be used subse%uently for
hybridization. &f prehybridization is inade%uate" the blot may have high background with
nonspecific probe binding.
'robe labeling is the ne(t step. There are several methods for labeling probes" but all
methods re%uire that the DNA molecule first be denatured to separate the )
complementary strands. This is usually performed by boiling the probe DNA for * min
then rapidly cooling. +apid cooling helps to prevent renaturation of the complementary
strands.
The most sensitive method of detection uses probes that are labeled with radioactive
#)
'
by random priming or nick translation. These methods add a
#)
' labeled nucleotide ,often
deo(y -T'. throughout the probe DNA molecule. The labeling reaction is then passed
through a column that binds unincorporated nucleotides and allows the radioactive DNA
to elute. This probe is very hot ,/(/$
0
cpm1g DNA. and can be used to detect single
copy genes with ease. The radioactive DNA that binds specifically to the probe is
detected by placing the membrane ne(t to 23ray film" or analyzing the membrane in a
phosphoimager. The use of
#)
' is also associated with potential hazards of e(ternal
contamination.
#)
' is a high3energy beta emitter and the researcher must take precautions
to shield the body from radiation. 4sually this is done by working behind a small
'le(iglass shield. All of the e(perimental waste material must be carefully retained and
then disposed" and careful records must be kept. All sources of
#)
' must be kept under
lock and key in the lab ,i.e. lab doors are always locked when using
#)
'. which can be an
inconvenience.
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' is rather e(pensive and has a short half3life. &n fact" probes must be
used within several days or they decay so much that they are no longer useful.
Another method for labeling probes is by utilizing chemiluminescent detection methods.
These are slightly less sensitive than
#)
' and are not widely used for Southern or Northern
blotting ,+NA detection. when sensitivity is important. 5owever" there is little or no
hazard associated with chemiluminscent probes and they have achieved wide use in
6estern blot applications. This method works by directly labeling the probe DNA with an
alkaline phosphatase enzyme. This is achieved by first denaturing the probe DNA and
then adding the enzyme along with a cross linking reagent. The alkaline phosphatase
labeled DNA that specifically binds to the complimentary DNA on the blot is detected by
placing the washed blot in a special substrate solution that alkaline phosphatase can
dephosphorylate. This reaction is associated with the release of chemiluminescence
which can be detected with 23ray film or a phosphoimager. !n the positive side" there is
no biological hazard. 7ou can leave the lab doors open" drink coke in the lab" and throw
the waste in the regular trash. The probes also have a long half3life and they can be stored
for weeks to months.
A third method of detection is colorimetric. This method is very similar to the
chemiluminescent protocol" e(cept that the sensitivity is much lower. The probe DNA is
labeled with an alkaline phosphatase enzyme by cross3linking and the labeled DNA
hybridizes specifically to the complementary gene on your membrane. The color
detection method uses a substrate for the alkaline phosphatase enzyme that becomes
insoluble and turns blue when it is cleaved. This method is inherently less sensitive than
chemiluminescence or radioactivity. 5owever" it has the advantage that it re%uires no
darkroom or film development reagents.
The third step is hybridization of the labeled probe to the membrane. This is usually
performed in a hybridization oven" which carefully regulates temperature and allows the
blot to turn constantly so that it is continually bathed in new hybridization solution. There
is definitely an optimal temperature for performing hybridization reactions. &f the
temperature is too low ,low stringency. the DNA strands can 8oin rather easily and they
often are able to 8oin even if the complimentary strands dont match completely. 9or
e(ample" at low stringency" a probe for the beta actin gene might cross hybridize with a
gene for alpha actin. The alpha actin gene from humans might hybridize to the alpha actin
gene from a dog. The match doesnt need to be perfect. This can be an advantage if one is
searching for new members of a gene family in the same species ,maybe you want to use
low or rela(ed stringency to look for new actin genes.. &t is also useful if you are
searching for a gene in another species that has not yet been cloned ,trying to find the
actin gene in the :ast African 7ellow ;ellied Snail Darter or some other obscure species.
!n the other hand" you can raise the temperature too high ,high stringency. so that it is
very difficult for any DNA to hybridize. This is because the temperature is too close to
the DNA melting temperature so the hybrids that form are easily denatured again. 4nder
these circumstances" only those se%uences that are perfectly matched can form stable
hybrids.
5ow do you choose the right temperature< &t depends on many factors including the =-
content of the DNA ,=- rich DNA melts differently than AT rich DNA." salt
concentration ,high salt means lower stringency." or the presence of formamide ,this
lowers the melting temp of DNA.. 7ou can also control for stringency of hybridization at
the ne(t step" which is washing the unbound probe away from the blot. &f you wash at
high temperature or low salt concentration" you will remove any hybrids that are not
perfectly matched. &f you wash at low temperature and1or in high salt" you are leaving
many imperfect hybrids on your membrane. 4sually" washing proceeds with a low
stringency wash at first to remove most of the unbound probe. This is followed by a
higher stringency wash. !ne advantage of using radioactive probes" is that you can easily
monitor how hot your blot is by simply checking it with the =eiger counter. &f it were too
hot you would use a more stringent wash. 6ith chemiluminescent or colorimetric
)
detection" you will not know whether you have too much background until you actually
develop he blot.
Objectives:
The ob8ective of this lab is to provide e(perience in labeling DNA probes and using the
probe in a hybridization reaction. The lecture will cover basic aspects of probe labeling
and hybridization.
Materials:
Alk'hos Direct >abeling and Detection ?it
5ybridization buffer
'rimary wash buffer ,)@ urea" $./A SDS" *$ m@ sodium phosphate at p5 B" /*$ m@
Sodium chloride" / m@ magnesium chloride" $.) A blocking reagent.
/$2 Secondary wash buffer ,/@ Tris p5 /$" )@ sodium chloride.
Alkaline 'hosphate -on8ugate Substrate ?it
5ybridization oven set at **C-
5ybridization bottles and nylon mesh
#BC- water bath
**C- water bath
Shaker platform
'lastic dishes for washing blots
'ipetters and yellow tips
&ce water bath
:ppendorf microcentrifuge
'reviously prepared blot with +NA sample
:ppendorf tubes
;oiling water bath
Dark room with developer and fi(er solutions
9ilm cassettes
23ray film
'lastic wrap
Methods:
Prehybridization:
/. 'reheat the re%uired volume of hybridization buffer to **C- in the hybridization
oven. 5eat enough buffer for $.)* ml1cm
)
of membrane. Also" preheat the glass
hybridization bottle containing /* ml of deionized water.
). +ehydrate the nitrocellulose membrane in water for *3/$ min.
#. 'lace the blot on a sheet of nylon mesh that is slightly larger ,/3) mm on each
side.. @ake sure that the DNA side of the blot is facing up. -arefully" roll the blot
and mesh into a roll and slip the roll into the glass hybridization bottle. The DNA
side should face into the hybridization chamber.
#
D. 'lace the cap on the tube and hold the tube horizontally. Turn the tube slowly until
the membrane unrolls inside the tube and is applied to the walls of the tube.
&nspect the tube and membrane carefully to make sure that there are no air
bubbles between the tube wall and the membrane. &f air bubbles are present" pull
the filter out and start again. Any air bubbles will lead to high background.
*. !nce the filter is applied to the wall of the tube" pour out the water and add about
/* ml of hybridization buffer. -ap the tube and place it into the clips in the
hybridization oven. Turn on the speed control such that the bottle turns slowly
through the oven ,one revolution every /$ sec.. -heck to see that the bottle is
even and that the hybridization fluid covers the bottom of the bottle.
E. Allow the blots to prehybridize ,before adding the probe. for appro(imately #$
min. This step is important to block any nonspecific reactive sites on the blot.
>ack of ade%uate prehybridization can lead to high background due to nonspecific
binding of probe to the membrane.
Prearation of robe:
B. 'repare the labeled hybridization probe. Dilute )$ l of cross linker solution with
0$ l of the water supplied with the kit. This working concentration should be
kept on ice.
0. Dilute 5'F3/E DNA to a concentration of /$ ng1l using the water supplied with
the kit.
G. 'lace /$ l of the diluted DNA sample into an eppendorf tube and denature the
DNA by heating for * min in a boiling water bath.
/$. &mmediately cool the DNA on ice for * min. ;riefly spin the sample in a
microcentrifuge to collect the contents at the bottom of the tube.
//. Add /$l of reaction buffer to the cooled DNA and mi( thoroughly but gently. ;e
sure to keep the tube on ice.
/). Add ) l of labeling reagent and mi( thoroughly but gently.
/#. Add /$ l of the cross linker working solution. @i( briefly and spin to collect the
contents at the bottom of the tube.
/D. &ncubate the reaction at #BC- for #$ min. The probe can be used immediately or
kept on ice for up to ) hours.
D
Hybridization reaction:
/*. Add labeled probe to the buffer used for prehybridization. 4se about *3/$ ng of
probe per ml of hybridization buffer. Avoid placing the probe directly on the blot.
+emove a small amount of and mi( with the probe before returning this mi(ture
to the bulk of the hybridization buffer.
/E. 5ybridize at **C- for ) days in the hybridization oven.
Post hybridization !ashes:
/B. 'reheat the primary wash buffer to **C- ,do not overheat.. 4se this in e(cess at a
volume of )3* ml per cm
)
of membrane.
/0. -arefully remove the roller bottle from the oven. This is easier if you briefly
switch off the motor for rotation" and then turn it on again after the bottle is
removed. 'our out the hybridization buffer from the roller bottle and add the
preheated primary wash buffer ,fill the tube halfway..
/G. 'lace the tube with wash buffer back in the hybridization oven and wash for /$
min at **C-.
)$. After /$ min" remove the roller bottle and pour out contents. Add more
prewarmed primary hybridization buffer and allow washing in the oven for
another /$ min at **C-.
)/. -arefully remove the membrane and mesh from the hybridization bottle using
forceps. 'lace the membrane in a plastic wash dish with /$$3)$$ ml of secondary
wash buffer. 6ash with gentle agitation on a shaker platform for /$ min at room
temperature. Several blots may be washed in the same secondary wash buffer
provided that there is enough volume to allow them to move freely.
)). 'our off the wash buffer and add /$$3)$$ ml of fresh secondary wash buffer.
Shake gently for an additional /$ min at room temperature.
"he#ilu#inescent signal detection:
)#. Allow the membrane to drain and briefly dab any e(cess fluid away with a paper
towel
)D. Add #ml of chemiluminescent substrate to your blot and allow it to saturate the
membrane. This can be done by placing the blot and substrate in an empty yellow
tip bo( and rocking back and forth.
*
)*. Dab e(cess substrate from blot using a paper towel and wrap the membrane
carefully in plastic wrap. Tape the wrapped membrane to the inside of an 23ray
film cassette.
)E. ;ring the cassette and an unopened 23ray film into the darkroom. Turn on the
safelight and shut the door. -heck to see that no light is coming in from outside.
)B. !pen the 23ray film and carefully place a sheet into the cassette so that it covers
the blot. -lose the cassette cover and allow the film to be e(posed for / hour.
@ake sure that the cassette snaps closed ,listen for the click..
)0. After e(posure" return to the darkroom and close the door. 6ear late( gloves.
Turn on the safelight and open the cassette. 'lace the film in the developer for
appro(imately #$ sec with periodic agitation. 7ou should see the image of the blot
appear.
)G. Transfer the developed film to water to remove e(cess developer and agitate for )
to # min.
#$. 'lace the film in fi(er and leave for ) to # min. After this" you can turn on the
light. After #$ min" you can place the film in water and wash for / to ) hours.
@ost modern molecular biology labs have an automated film processor that
automatically develops films in / to ) min.
#/. -lean upH
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