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Northern & Southern Northern & Southern HybridizationHybridization

Avalon Garcia

02-20-04

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IntroductionIntroduction Concept: reannealing nucleic acids to identify

sequence of interest.

Separates DNA/RNA in an agarose gel, then detects specific bands using probe and hybridization.

Hybridization takes advantage of the ability of a single stranded DNA or RNA molecule to find its complement, even in the presence of large amounts of unrelated DNA.

Allows detection of specific bands (DNA fragments or RNA molecules) that have complementary sequence to the probe.

Size bands and quantify abundance of molecule.

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Southern Blot: DNA-DNA*Southern Blot: DNA-DNA*

Developed by Edwin Southern.Developed by Edwin Southern.

Uses gel electrophoresis together with Uses gel electrophoresis together with hybridization probes to characterize restriction hybridization probes to characterize restriction fragments of genomic DNA (or DNA from other fragments of genomic DNA (or DNA from other sources, such as plasmids). sources, such as plasmids).

Identifies DNA with a specific base sequence.Identifies DNA with a specific base sequence.

Can be done to detect specific genes present in Can be done to detect specific genes present in cells. cells.

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Southern StepsSouthern Steps1.1. DNA to be analyzed is digested to completion

with a restriction endonuclease.

2. Electrophoresis to maximally separate restriction fragments in the expected size range. A set of standards of known size is run in one lane of the gel.

3. Blot fragments onto a nitrocellulose membrane.

4. Hybridize with the 32P probe.

5. Autoradiography.

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Step 2Step 2

Gel electrophoresisGel electrophoresis Separates DNA Separates DNA

fragments.fragments.

Soak gel in 0.5 M NaOHSoak gel in 0.5 M NaOH Converts dsDNA to Converts dsDNA to

ssDNAssDNA

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Step 3. Nitrocellulose Step 3. Nitrocellulose BlotBlot

Cover gel with Cover gel with nitrocellulose paper…nitrocellulose paper…then…then…

Cover nitrocellulose paper Cover nitrocellulose paper with thick layer of paper with thick layer of paper towels.towels.

Compress apparatus with Compress apparatus with heavy weight.heavy weight.

ssDNA binds to ssDNA binds to nitrocellulose at same nitrocellulose at same position it had on the gel.position it had on the gel.

Vacum dry nitrocellulose Vacum dry nitrocellulose at 80at 80C to permanently fix C to permanently fix DNA in place or cross link DNA in place or cross link (via covalent bonds) the (via covalent bonds) the DNA to the membrane. DNA to the membrane.

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Step 4. HybridizationStep 4. Hybridization Incubate nitrocellulose Incubate nitrocellulose

sheet with a minimal sheet with a minimal quantity of solution quantity of solution containing containing 3232P-labeled P-labeled ssDNA probe.ssDNA probe.

Probe sequence is Probe sequence is complementary to the DNA complementary to the DNA of interest.of interest.

Incubate for several hours Incubate for several hours at suitable renaturation at suitable renaturation temperature that will temperature that will permit probe to anneal to permit probe to anneal to its target sequence(s).its target sequence(s).

Wash & dry nitrocellulose Wash & dry nitrocellulose sheet.sheet.

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Step 5. AutoradiographyStep 5. Autoradiography

Place nitrocellulose sheet Place nitrocellulose sheet over X-ray film.over X-ray film.

X-ray film darkens where X-ray film darkens where the fragments are the fragments are complementary to the complementary to the radioactive probes.radioactive probes.

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General Scheme for Southern BlotGeneral Scheme for Southern Blot

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Southern Application: Southern Application: Diagnosis & detection of genetic diseases.Diagnosis & detection of genetic diseases.

Used to diagnose sickle cell-anemia.Used to diagnose sickle cell-anemia.

AAT base change in the T base change in the subunit of Hb subunit of Hb

GluGlu Val. Val.

Development of a 19 residue oligonucleotide Development of a 19 residue oligonucleotide probe complementary to sickle-cell gene’s probe complementary to sickle-cell gene’s mutated segment.mutated segment.

Probe hybridizes to DNA from homozygotes of Probe hybridizes to DNA from homozygotes of sickle-cell anemia but not from normal individuals.sickle-cell anemia but not from normal individuals.

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Northern Blot: RNA-Northern Blot: RNA-DNA*(RNA*)DNA*(RNA*)

Alwine adapted Southern's method for DNA to detect, size and quantify RNA – 1977.

No need to digest RNA with restriction enzymes.

Use formaldehyde to break H-bonds and denature RNA because single-stranded RNA will form intramolecular base pairs and "fold" on itself.

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Northern StepsNorthern Steps1.1. Isolate RNA & treat with formaldehyde.Isolate RNA & treat with formaldehyde.

2.2. Electrophorese RNA in denaturing agarose gel Electrophorese RNA in denaturing agarose gel (has formaldehyde). Visualize RNA in gel using (has formaldehyde). Visualize RNA in gel using Ethidium bromide stain and photograph.Ethidium bromide stain and photograph.

3.3. Transfer single-stranded RNA to nitrocellulose or Transfer single-stranded RNA to nitrocellulose or nylon membrane. Covalently link RNA to nylon membrane. Covalently link RNA to membrane.membrane.

4.4. Incubate membrane (RNA immobilized on Incubate membrane (RNA immobilized on membrane) with labeled DNA or RNA probe with membrane) with labeled DNA or RNA probe with target sequence.target sequence.

5.5. Development.Development.

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Step 1Step 1Isolate RNA:Isolate RNA:

-To detect rare mRNA, isolate the poly A-To detect rare mRNA, isolate the poly A++ mRNA. mRNA.

-RNA is both biologically and chemically more -RNA is both biologically and chemically more labile than DNA. Thus eliminate RNases.labile than DNA. Thus eliminate RNases.

Step 2Step 2Electrophoresis:Electrophoresis:

-Performed in formaldehyde agarose gel to prevent -Performed in formaldehyde agarose gel to prevent RNA from folding on itself.RNA from folding on itself.

-Stain with EtBr to visualize the RNA bands.-Stain with EtBr to visualize the RNA bands.

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Step 3Step 3-Transfer single-stranded RNA to nitrocellulose -Transfer single-stranded RNA to nitrocellulose

or nylon membrane:or nylon membrane:

Traditionally, a nitrocellulose membrane is Traditionally, a nitrocellulose membrane is used, although nylon or a positively charged used, although nylon or a positively charged nylon membrane may be used. nylon membrane may be used.

Nitrocellulose typically has a binding capacity Nitrocellulose typically has a binding capacity of about 100µg/cm, while nylon has a binding of about 100µg/cm, while nylon has a binding capacity of about 500 µg/cm. Many scientists capacity of about 500 µg/cm. Many scientists feel nylon is better since it binds more and is feel nylon is better since it binds more and is less fragile. less fragile.

-Covalently link RNA to membrane:-Covalently link RNA to membrane:

UV cross linking is more effective in binding UV cross linking is more effective in binding RNA to the membrane than baking at 80RNA to the membrane than baking at 80C.C.

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Step 4 & 5Step 4 & 5-Prehybridize before hybridization:-Prehybridize before hybridization:

Blocks non-specific sites to prevent the single-Blocks non-specific sites to prevent the single-stranded probe from binding just anywhere on stranded probe from binding just anywhere on the membrane. the membrane.

-Incubate membrane with labeled DNA or RNA -Incubate membrane with labeled DNA or RNA probe with target sequence:probe with target sequence:Probe could be Probe could be 3232P, biotin/streptavidin or a P, biotin/streptavidin or a bioluminescent probe. bioluminescent probe.

-Autoradiography:-Autoradiography:

Place membrane over X-ray film.Place membrane over X-ray film.X-ray film darkens where the fragments are X-ray film darkens where the fragments are complementary to the radioactive probes.complementary to the radioactive probes.

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Agarose gel & Membrane & Agarose gel & Membrane & AutoradiographyAutoradiography

Sobel, R., Dubitsky, A., Brickman, Y. (2002) Genetic Engineering News 23, 2.

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Northern ApplicationNorthern Application

Northern blots are particularly useful for determining the conditions under which specific genes are being expressed, including which tissues in a complex organism express which of its genes at the mRNA level.

For instance:

When trying to learn about the function of a certain protein, it is sometimes useful to purify mRNA from many different tissues or cell types and then prepare a Northern blot of those mRNAs, using a cDNA clone of the protein of interest as the probe. 

Only mRNA from the cell types that are synthesizing the protein will hybridize to the probe.

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SummarySummary

SouthernSouthern DNA on membrane.DNA on membrane. Digest DNA.Digest DNA. Convert dsDNA to Convert dsDNA to

ssDNA.ssDNA. Probe with DNA or Probe with DNA or

RNA.RNA.

NorthernNorthern RNA on membrane.RNA on membrane. No need to digest No need to digest

DNA.DNA. Denature “folded” Denature “folded”

RNA with RNA with formaldehyde.formaldehyde.

Probe with DNA or Probe with DNA or RNA.RNA.

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ReferencesReferencesKornberg, A., Baker, T. A., DNA Replication. (2nd ed.) pp 15, W. H.

Freeman & Co. (1992)

Sobel, R., Dubitsky, A., Brickman, Y. (2002), Genetic Engineering News, 23, 2.

Southern, E. M., (1975) J. Mol. Biol. 98:503-517.

Voet, D., Voet, J. Biochemistry. (vol. 1, 3rd ed.), pp 110-112, John Wiley & Sons Inc. (2004).

http://homepage.smc.edu/colavito_mary/biology22/2www.colorado.edu/MCDB/MCDB2150Fall/notes99/99L17.html

www.uwp.edu/~higgs/Lect12mb.htmlwww.uwp.edu/~higgs/Lect12mb.html

www.uwp.edu/~higgs/Lect13mb.htmlwww.uwp.edu/~higgs/Lect13mb.html

www.sou.edu/biology/courses/bi342/Lect5.htmwww.sou.edu/biology/courses/bi342/Lect5.htm

www.med.unc.edu/pmbb/MBT2000/Northern%20Blotting.htmwww.med.unc.edu/pmbb/MBT2000/Northern%20Blotting.htm