Genomic Southern Genomic Southern BlottingBlotting

and and DNA FingerprintingDNA Fingerprinting

ABE Workshop 2007ABE Workshop 2007

Jamie LumJamie Lum

Southern Blotting is a Southern Blotting is a method to check for the method to check for the presence of a gene or presence of a gene or DNA in a sample. DNA in a sample.

Group 1Group 1 Group 2Group 2 Group 3Group 3 Group 4Group 4

PDI-2PDI-2 PDI-2PDI-2 PDI-3PDI-3 PDI-3PDI-3

Bam HIBam HI Hind IIIHind III Bam HIBam HI Hind IIIHind III

1kB WT HM HZ LD 1kb WT HM HZ LD 1kB WT HM HZ LD 1kB WT HM HZ LD1kB WT HM HZ LD 1kb WT HM HZ LD 1kB WT HM HZ LD 1kB WT HM HZ LD

Mark U C U C U C Mark C U C U C U Mark C U C U C U Mark U C U C U C Mark U C U C U C Mark C U C U C U Mark C U C U C U Mark U C U C U C

Quantitate DNA with serial Quantitate DNA with serial dilutionsdilutions

In the presence of In the presence of NBT/BCIP, alkaline NBT/BCIP, alkaline phosphatase (AP) phosphatase (AP) precipitates into precipitates into purple spots.purple spots.

1010-1-1 10 10-2-2 10 10-3 -3

1010-4-4

ControlControl

Group 1Group 1Group 2Group 2Group 3Group 3Group 4Group 4

Capillary transfer of the DNA Capillary transfer of the DNA fromfrom

the gel to a nylon membranethe gel to a nylon membrane Cut off top right corner Cut off top right corner

of the gel.of the gel. Immerse gel in Immerse gel in

0.125M HCl and shake 0.125M HCl and shake for 10 minutes.for 10 minutes.

Soak the gel in Soak the gel in Denaturation Buffer Denaturation Buffer for 30 minutes on for 30 minutes on shaker.shaker.

Rinse the gel with Rinse the gel with water.water.

Soak in Neutralization Soak in Neutralization Buffer for 30 minutes.Buffer for 30 minutes.

• Cut nylon membrane Cut nylon membrane and two 3M filter and two 3M filter papers to the size of the papers to the size of the gel. Cut off top right gel. Cut off top right corner of membrane.corner of membrane.

• Add transfer buffer to a Add transfer buffer to a glass tub.glass tub.

•LAYER, LAYER, LAYER, AND LAYER, AND LAYER!!LAYER!!

• Tray with transfer Tray with transfer bufferbuffer

• Wick (3M paper)Wick (3M paper)• GelGel• NitrocelluloseNitrocellulose• Parafilm cut as a Parafilm cut as a

picture framepicture frame• Filter paperFilter paper• Paper towelsPaper towels• Glass dishGlass dish• WeightWeight• Allow to sit Allow to sit

overnightovernight

Mark gel wells on the membrane with a Mark gel wells on the membrane with a pencil.pencil.

Rinse the membrane in 6X SSC for 10 Rinse the membrane in 6X SSC for 10 minutes.minutes.

Lay the membrane, nucleic acid side Lay the membrane, nucleic acid side up, on a piece of foil and expose to up, on a piece of foil and expose to 120,000 microjoules/cm120,000 microjoules/cm22..

PrehybridizatioPrehybridization andn and

HybridizationHybridization

• Add 10 mL of 42Add 10 mL of 42ooC DIG Easy Hyb C DIG Easy Hyb

solution to a roller tube with the solution to a roller tube with the membrane.membrane.

• Prehybridize for 20-30 minutes in a Prehybridize for 20-30 minutes in a 4242ooC hybridization oven.C hybridization oven.

• Denature probe in boiling water for 5 Denature probe in boiling water for 5 minutes and quickly place in ice water.minutes and quickly place in ice water.

• Remove prehybridization solution and Remove prehybridization solution and add probe/hybridization mixture into add probe/hybridization mixture into roller tube.roller tube.

• Hybridize for at least 2 hours in the Hybridize for at least 2 hours in the hybridization oven.hybridization oven.

• Pour off the probe.Pour off the probe.

• Wash membrane twice in 2X Wash solution Wash membrane twice in 2X Wash solution

for 5 minutes per wash.for 5 minutes per wash.• Wash membrane twice in 0.1X Wash Wash membrane twice in 0.1X Wash

solution for 10 minutes per wash.solution for 10 minutes per wash.• Equilibrate membrane in Washing Buffer for Equilibrate membrane in Washing Buffer for

2 minutes.2 minutes.• Incubate in Blocking solution for 30 minutes.Incubate in Blocking solution for 30 minutes.• Remove blocking solution and add Antibody Remove blocking solution and add Antibody

solution. Incubate for 30 minutes.solution. Incubate for 30 minutes.• Wash membrane twice in Washing Buffer for Wash membrane twice in Washing Buffer for

15 minutes per wash.15 minutes per wash.• Equilibrate membrane in Detection Buffer Equilibrate membrane in Detection Buffer

for 2 minutes.for 2 minutes.

• Place membrane on a piece of Place membrane on a piece of

transparency film and spot with transparency film and spot with CSPD/Detection Buffer over membrane.CSPD/Detection Buffer over membrane.

• Wrap up edges, make sure no air bubbles.Wrap up edges, make sure no air bubbles.• Tape membrane in pre-warmed X-ray Tape membrane in pre-warmed X-ray

cassette and incubate for 10 minutes.cassette and incubate for 10 minutes.• In dark room, place cut X-ray film over In dark room, place cut X-ray film over

membrane and close cassette.membrane and close cassette.• Expose the film for 30 minutes.Expose the film for 30 minutes.• Remove film from cassette and place in Remove film from cassette and place in

developer for 1 minute.developer for 1 minute.• Transfer film to fixer for 5 minutes.Transfer film to fixer for 5 minutes.• Rinse with tap water and hang to dry.Rinse with tap water and hang to dry.

F F IINNAALLLLYY!!!!

Group 1Group 1MW U C U C U C LamHindIII

WT HMPDI2 HZPDI2

Group 2Group 2MW C U C U C U LamHindIII

WT HMPDI2 HZPDI2

Group 3Group 3

MW C U C U C U LamHindIII

WT HMPDI3 HZPDI3

Group 4Group 4MW U C U C U C LamHindIII

WT HMPDI3 HZPDI3

So how is this information So how is this information useful?useful?

• Restriction Fragment Length Restriction Fragment Length Polymorphisms or RFLPs differentiates by Polymorphisms or RFLPs differentiates by patterns derived from cleavage of their patterns derived from cleavage of their DNA. The similarity of the patterns DNA. The similarity of the patterns generated by RFLPs can be used to generated by RFLPs can be used to differentiate species or individuals from differentiate species or individuals from one another. one another.

• Isolation of DNA for analysis is both time Isolation of DNA for analysis is both time consuming and labor intensive.consuming and labor intensive.

• Polymerase Chain Reaction or PCR is a Polymerase Chain Reaction or PCR is a technique for amplifying a specific technique for amplifying a specific region of DNA, defined by a set of two region of DNA, defined by a set of two "primers" at which DNA synthesis is "primers" at which DNA synthesis is initiated by a thermostable DNA initiated by a thermostable DNA polymerase. polymerase.

• Used to amplify small amounts of DNA Used to amplify small amounts of DNA in a few hours so more samples can be in a few hours so more samples can be analyzed in a shorter time.analyzed in a shorter time.

• Paternity testingPaternity testing

• Positive identification of crime suspectPositive identification of crime suspect

Someone ate the last cookie. A small DNA Someone ate the last cookie. A small DNA

sample was taken from the plate and DNA sample was taken from the plate and DNA was taken from seven of the workshop was taken from seven of the workshop facilitators. Based on the RFLP, who did it?facilitators. Based on the RFLP, who did it?

1-Dr. Christopher1-Dr. Christopher

2-Dong Ping2-Dong Ping

3-Prof. Kabi3-Prof. Kabi

CS-Crime Scene DNACS-Crime Scene DNA

4-Eun Ju4-Eun Ju

5-Dr. White5-Dr. White

6-Dr. Unabia6-Dr. Unabia

7-Dr. Berestecky7-Dr. Berestecky

1 2 3 CS 4 5 6 71 2 3 CS 4 5 6 7

A woman has accused a A woman has accused a

man of fathering her man of fathering her twins. Of course, he twins. Of course, he denies it, so a paternity denies it, so a paternity test was done.test was done.

1-Mother1-Mother

2-Child 12-Child 1

3-Child 23-Child 2

4-Potential father4-Potential father

1 2 3 4 1 2 3 4