Hybridization Prof Dr Naggar

7/31/2019 Hybridization Prof Dr Naggar

1/32


2/32

Hybridization with radiolabeledoligonucleotide probes to nucleic acids

immobilized on nylon membrane:

(Sambrook et al., 1989).


3/32


4/32


5/32

Southern, E.M. (1975): Detection ofspecific sequences among DNA

fragments separated by gel

electrophoresis. J. Mol. Biol., 98 : 503.


6/32


7/32


8/32


9/32


10/32


11/32


12/32


13/32


14/32

Methods of transfer of DNA:

Capillary transfer.

Draws the buffer up by capillary action through the gel

into the membrane, which will bind ssDNA.

Electrophoretic transfer.

Vacuum transfer.

A vacuum sucks SSC through the membrane. This workssimilarly to capillary action, excepts more SSC goes

through the gel and membrane, so it is faster (about an

hour).


15/32


16/32


17/32


18/32

Oligonucleotides as short as 17 nucleotidesmay be used.

Because of small size of target sequence, aminimum of 30 g of genomic DNA shouldbe applied to each lane.


19/32

Reagents :Hybridization solution:

In experiments designed tovalidate the PCR products,antisense 30-mer oligonucleotide

probes internal to the amplifiedfragments were synthesized.


20/32

Synthetic oligonucleotides weresynthesized without a phosphate group at

their 5' termini and were therefore easilylabeled by transfer of -32p from [ -32p]ATP using the enzyme bacteriophage T4polynucleotide kinase.

100 ng of each probe were end-labeled with25 uCi of ] ATP (sp act 3000 Ci/mmol)using 10 U of T4 polynucleotide kinase (10U/ml )in the presence of 70 mM Tris-HCl(pH 7.6), 10 mM MgCl2 and 5 mMditiothretiol in a final volume of 20 l.


21/32

The smaller the volume of the hybridizationsolution, the better.

In small volumes, the kinetics of nucleic acid

reassociation are faster and the amount ofprobe can be reduced.

However it is essential that sufficient liquidbepresent for the filter to remain covered all timesby a film of the hybridization solution.


22/32

To minimize background problems, it isbest to hybridize for the shortest timeusing minimum amount of probe.

For mammalian genomic DNA whereeach lane of the gel contains 10 g ofDNA, 10-20 ng/ml radiolabeled probe

should be used.


23/32

Prehybridization solution:

The nylon membranes wereprehybridized in 30 ml of buffer

composed of:- 0.25% Sodium dodecyl sulfate (SDS)

- 1.2x SSC

- 2x Denhardt's reagent

- heat-denatured salmon sperm DNA (10mg/ml) .


24/32

Denhardt's reagent:

It is usually made up as a 50x stocksolution, which is filtered and stored at

-20 C.50x Denhardt's reagent contains 5 gmFicoll (type 400, Pharmacia), 5 gm of

polyvinylpyrrolidone, 5 gm of bovineserum albumin (Fraction V, Sigma) anddistilled water to 500 ml.


25/32

Denhardt reagent is used to block thenonspecific attachment of the probe tothe surface of the filter.

Frequently, it is used in combinationwith heat-denatured salmon spermDNA and detergents such as SDS and

this leads to complete suppression ofbackground.

d d lH


26/32

denatured salmon-Heat

sperm DNA:Salmon sperm DNA (Sigma type III sodium salt)was dissolved in distilled water at a concentrationof 10 mg/ml.

The concentration of NaCl was adjusted to 0.1 Mand the solution was extracted once with phenoland once with phenol:chloroform.

The aqueous phase was removed , and the DNAwas sheared by passing it 12 times rapidlythrough a 17-gauge hypodermic needle.The DNA was precipitated by adding 2 volumes of

ice-cold ethanol.

d d lH


27/32

denatured salmon-Heat

sperm DNA:It was then recovered by centrifugationand redissolved at a concentration of 10mg/ml in water.The OD 260 of the solution wasdetermined and the exact concentration ofthe DNA was calculated.

The solution was then boiled for 10 min.and stored at -20 oC in small aliquots .Just before use, the solution was heatedfor 5 min. in a boiling-water bath and then

chilled quickly in ice water.


28/32

Steps :

The nylon membrane was put into a plastic traycontaining 30 ml of prehybridization solution and

allowed to wet first from beneath then submergedand incubated for 1 hour at 45 oC with gentleagitation.

0.2 ml of prehybridization solution will be requiredfor each square centimeter of nylon membrane.

The radiolabeled probe was added to the

prehybridization solution and the membrane wasincubated for 2 hours at 45oC with gentleagitation.


29/32

The hybridization solution was poured outinto a container suitable for disposal andthe membrane was immediately submergedin a tray containing 100 ml of 2x SSC . 0.05%

SDS and washed at 52oC for 5 min. withrapid agitation.

the washing solution was poured andwashing was repeated at 52oC for another30 min.


30/32

(sometimes washing may be repeateddepending on the clearance of thebackground after developing X ray film,

however excessive washing was avoided asit may remove the weak bands from themembrane).

Montor the radioactivity on the filter using ahand-held minimonitor.


31/32

The wet membrane was placed on a

sheet of saran wrap and covered with asecond sheet of saran wrap, andexposed to X ray film in a tight X ray

shield for 2 - 4 hours at - 70oC.

then the film was developed to obtainan autoradiographic image .


32/32

Intensities of bands inautoradiographs were quantitatedusing a densitometer and results

were expressed as percentages of -actin mRNA copies .

Documents

Hybridization Prof Dr Naggar