HYBRIDIZATIONMETHODS

Week 12

• Detection ofdisease agents inmaterials (tissue,organ,cell culture,excretion,secretion,etc.)or specific genes incell DNAofthese tissueswith labeled probes• Probe hybridization methods

1. Southern Blot Hybridization2. Northern Blot Hybridization3. Dot Blot Hybridization4. In situ Hybridization

Southern Blot Hybridization

1. Isolation ofgenomic DNA2. Cutting with specific restriction endonuclease (RE)enzyme3. Electrophoresis offragments4. Transferofthe bands onthe membrane (Southern transfer)5. Denaturation and fixation6. Hybridization7. Autoradiography

• Developed by Southern• Specific sequences inisolated and then purified DNAofm.o.found indiagnostic materials are revealed with the help oflabeled probes(DNAor RNAprobe).

1.Isolation ofgenomic DNA

• Cellwalls and outer membranes ofm.o.are lysed using lysozyme andother enzymes.• Componentsother than DNAare removed by adding SDS(sodiumdodecyl sulfate),proteinase Kand Rnase to the extraction solution.• EDTAisadded to protect DNAfromendogenous nuclease.• Phenol chloroformextraction ineukaryotes• Cesium chloride density gradient centrifugation to removepolysaccharides

• The concentration ofDNA;spectrometric or electrophoretic methods• Tissue or organcell DNAsuitable extraction method• Genetic materials belonging to plasmids,phages and viruses,withappropriatemethods ...

2.Cutting with specific RE

• Enzymes like EcoRI,Hind III,BamHI• One or two or more enzymes inthe experiment• 4-6hours at37C• Alarge number ofDNAsegments,small and large,by cutting DNAfrom certain bases

3.Electrophoresis ofFragments

• Electrophoresis inagarose gelat40-50Vovernight• Fragments are ordered indescending order• Bands formed onthe gelcanbestained with ethidium bromide andvisible under UV-rays.

• Nearly 100bands larger than 20kb inagood sectionIn revealing thedifferences inDNAs• RFLP,RAPDtechniques inmaking restriction analysis• PAGEisused alongside agarose gel

4.Transferofthe bands onthemembrane (Southern transfer)

• Transferofbands onagarose gelonnylon or nitrous cellulosemembrane• Electrotransfer,alkalinecapillarity• Nylon membranes are preferred.

5.Denaturation and Fixation

• DNAbands transferred onto nylon are dried• Denatured with 0.5NNaOH• It isheated up to 80C,single strands are fixed onthe membrane.• Fixation process isdoneinvacuum to secure fixation

6.Hybridization

• 2-4hours prehybridization at60-65C• Washing• Labeled DNAprobe (32Por biotin)or RNAprobe addition• Single-stranded and labeled probe onthe membrane are mutuallyjoined by non-covalent bonds with the single-stranded DNAsegmentthat isself-complementary and the double-stranded DNAxDNAduplex isformed (hybridization).• One night• Washing

7.Autoradiography• Afilmsensitive to X-rays iscovered onthe nylonmembrane.• It iskept at70Cfor 1night.Bath ofthe film• Regions with target DNAsequences combined withlabeled probes appear inblack.These regions refer tothe places between which the geneislocalized.• In biotin-labeled probes,the color change caused byavidin,peroxidase and chromogen substanceindicates the location ofthe target DNAsegment tobesought

Northern Blot Hybridization

• Instead ofDNA,mRNA,viral RNAor totalRNA• Separation ofRNAongel,transferto filter,hybridization with specificprobe (RNAor DNA)and autoradiography Northern Blot Hybridization• It isused to analyze genes atthe transcription level,to determine thelevel ofmRNA intissues and organs,and to detect the differencebetween viral RNAs.

Northern Blot Hybridization

1. Isolation ofRNA2. Separation ofRNAby electrophoresis3. TransferofRNAonto nylon membrane (Northern transfer)4. Hybridization with labeled DNAor RNAprobesImaging by

autoradiography

• RNAs are separated from the DNAby selectively precipitation withlithium chloride or sodium acetate by precipitation method.• NoREcutting required before separation ofRNAmolecules inagarosegelIf desired,• Southern blotting canbeperformed by convertingmRNA or viral RNAinto cDNA with reverse transcriptase enzyme.

3.Dot Blot Hybridization• Nucleic acids belonging to the causative agent areextracted frompathological materials andconcentrated.• Samples are diluted x2or x5Onedrop ofeach dilutionisapplied to the nylon or nitrocellulose membrane.• Fixed onthe membrane by denaturationSpecificsingle strand probemarked with 32Pisadded• Next transactions are asinSouthern Blot• Darkspots onfilmindicate hybridization zone

4.In Situ Hybridization

• Infected tissue cultures,biopsy materials,tissue suspensions aretaken onadrop ofperipheral blood onaslide.• After denaturation,alabeled (32P,biotin-avidin)• cDNA probe isadded and hybridization isachieved.• The location ofM.o.'s DNAiseasily determined.

DETERMINATIONOFIMMUNOGENICSUBSTANCES

• Biotechnological methods are used to reveal disease factors andantigenic substances (protein,glycoprotein,etc.)obtained fromthem.

1. Immuno (Western)Blotting2. ProteinDot Blotting Experiment3. In Situ Immunoperoxidase Assay4. UsingMonoclonal Antibodies

Immuno(Western)Blotting

• Proteins belonging to the agents are obtained inpure formandtreated with SDSand then separated according to their molecularweights by SDS-polyacrylamide gelelectrophoresis.• Transferred to nitrouscellulose papers by electrotransfer and air dried• The paper strips are treated with polyclonal or monoclonal antibodiesto allow the antibodies to bind with proteins (antigen).

• Washing• Goat anti-IgG specific antibodies prepared against the first antibodiesand labeled (32P,biotin)are placed onthe washed paper strips.• If 32Pmarked abs are used,the result isautoradiography,if biotiny abisused,avidin-peroxidase conjugate 4-chloro-1-naphtolsubstrate isadded to evaluate according to the color change.

ProteinDot Blot Assay• Specific monoclonal abs against the target agent areputonnitrocellulose papers and 10-15min.Dried• Supernatants ofmicroorganism suspensions (proteinantigen)prepared from infected tissues are drippedonto the antibodies dried onfilter paper.• 2hours standby (ab-ag conjugate)• The filter iswashed,antiserum prepared against theagent and labeled with biotin isdroppedonto theconjugate and incubated for 2hours and washed.• Avidin enzyme conjugate and substrate are added.

In Situ Immunoperoxidase Assay

• By using polyclonal or monoclonal antibodies,bacterial or viralproteins are detected informalin-fixed infected tissue sections.Sections are incubated with specific ab,aminoethyl-carbazole isadded assubstrate.Microbial proteins intissues are detectedaccording to color change.

Use ofMonoclonal Antibodies

• Fluorescein,peroxidase-antiperoxidase,biotin-avidin,radioisotopes,enzymes,etc.Monoclonal antibodies marked with are usedsuccessfully.