biotechnology and genetic engineering-pbio 450/ · ppt file · web...

MCB 7300: Southern Blot Lab• Southern, Northern & Western Blotting• Background• Overview• Detailed Protocol

Who is this guy?

Sir Edwin Mellor Southern of course (1938- )

• Inventor of the Southern blot • http://en.wikipedia.org/wiki/Edwin_Southern

Characterization: Southern blot hybridization

-transfer of DNA from a gel to a membrane (e.g., nitrocellulose, nylon)-developed by Edwin Southern

Characterization: Northern blot hybridization

-transfer of RNA from a gel to a membrane (e.g., nitrocellulose, nylon)-reveals mRNA size (and approximate protein size), tissue- and organ-specific expression, and kinetic patterns of expression

X RNA

X

xsalt

XRNA

Characterization: Western blotting

-transfer of protein from a gel to a membrane (e.g., nitrocellulose, nylon)-requires the use of an electric current to facilitate transfer

X Protein

X

x Buffer; requires electric current

X

React withAntibody

X

Enzymereaction

or

DNA Microarrays:An Introduction

MicroarrayResult:

Much analysis to follow

Southern blot hybridization

-transfer of DNA from a gel to a membrane (e.g., nitrocellulose, nylon)-developed by Edwin Southern

Southern blotting of genomic DNA Steps

1. Extraction of genomic DNA

2. Electrophoresis of genomic DNA (or more commonly restriction enzyme-digested genomic DNA)

3. Before blotting, treat the gel with 0.2N HCl, denaturation and neutralization solution

4. Blotting- Transfer gel to the nitrocellulose membrane by capillary action

A) Sponge method

B) Wick method

5. After the transfer, cross-link the DNA to the nitrocellulose membrane

A. UV- cross linking B. Bake at 120°C for 30 min C. Bake at 80°C for 2 hrs

6. Making the probe Label the probe to be hybridized using radioactive or

non-radioactive methods

Non-radioactive methods A) Colorimetric B) Chemiluminescent

* Roche DIG- DNA labeling (Non-radioactive) kits are used for detection

DIG or Digoxigenin is used for labeling the probe. DIG steroid found exclusively in the flowers and leaves of the plants Digitalis purpurea, Digitalis orientalis and Digitalis lanata (foxgloves).

(see http://en.wikipedia.org/wiki/Digoxigenin)

DIG-11-dUTP replaces dTTP in the random primed DNA labeling reaction.

DIG-DNA Labeling: DNA is random primed labeled with Digoxigenin-11-dUTP using DIG-High Prime, a 5x concentrated labeling mixture of random hexamers, dNTP mix containing alkali-labile Digoxigenin-11-dUTP, labeling grade Klenow enzyme and an optimized reaction buffer.

Structure of digoxigenin- and biotin-modified nucleotides Note that the digoxigenin and biotin groups in these examples are linked to the 5′ carbon atom of the uridine of dUTP by spacer groups consisting respectively of a total of 11 carbon atoms (digoxigenin-11-UTP) or 16 carbon atoms (biotin-16-dUTP). The digoxigenin and biotin groups are reporter groups: after incorporation into a nucleic acid they are bound by specific ligands containing an attached marker such as a fluorophore.

Arabidopsis T-DNA mutant

T-DNA

Gene

RBLB

T-DNA

Disrupted gene

RBLB1000bp probe

7. Prehybridization and hybridization of probe to the membrane at the hybridization temperature.

8. Washes and immunological detection

Detection after stringency washes using anti-digoxigenin antibody conjugated to AP (Alkaline phosphatase)

BCIP is colorless and is dephosphorylated by AP

Dephosphorylated BCIP is oxidized by NBT

Oxidized BCIP will turn into a dark blue indigo dye

NBT is reduced to a dark blue dye

Colorimetric detection using NBT/ BCIP

• 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) is a chemical compound used in immunoblotting, in situ hybridization, and immunohistochemistry, with nitro blue tetrazolium chloride (NBT), for sensitive colorimetric detection of alkaline phosphatase. NBT serves as the oxidant (and gives also dark blue dye) and BCIP is the alkaline phosphatase substrate. Alkaline phosphatase is commonly conjugated to secondary antibodies.

• BCIP (colorless)oxidation→ blue precipitate. BCIP-NBT naturally forms this bluish purple precipitate over time; however, alkaline phosphatase speeds up the process 1000 fold. BCIP binds very tightly in the alkaline phosphatase active site, but when NBT reacts with BCIP, it is released from the enzyme and the colored precipitate forms.

• Nitro blue tetrazolium is a chemical compound composed of two tetrazole moieties. It is used in immunology for sensitive detection of alkaline phosphatase (with BCIP). NBT serves as the oxidant and BCIP is the AP-substrate (and gives also dark blue dye). In immunohistochemistry the alkaline phosphatase is often used as a marker, conjugated to an antibody. The colored product can either be of the NBT/BCIP reaction reveals where the antibody is bound, or can be used in immunofluorescence.

BCIP and NBT: additional information

Colorimetric blot

Chemiluminescent detection using CSPD

• Dephosphorylation of CSPD by alkaline phosphatase to the metastable phenolate anion.

• Phenolate anion decomposes and emits light at a wavelength of 477 nm.

• Light emission is captured on X-rays.

• chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD), formally disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate is a chemical substance with formula C18H20ClO7PNa2. It is a component of enhanced chemiluminescence enzyme-linked immunosorbent assay (ELISA) kits, used for the detection of minute amounts of various substances.

• In typical uses of ELISA kits, the enzyme alkaline phosphatase removes the phosphate group from CSPD, yielding a reactive anion (phenolate, which then splits itself in two components, adamantane and 1,2-dioxetane. This second reaction emits turquoise-coloured light (λmax = 477 nm). The decomposition of the dioxetane generates a secondary glow.

CSPD reaction

Lab Report• The lab report counts 100 points and is due at the beginning

of lab on Wednesday, March 26, 2014.• Each student must prepare and submit a hard copy of their

lab report.• Your lab report should describe everything that was done in

the style of a PNAS paper.• It should include the following sections:• Abstract• Introduction (on the method & objectives & gene amplified)• Results • Discussion• Materials and methods (summarized protocol)• Acknowledgements• References • Answers to the 10 questions on the next slide.

Questions for your lab report1. Explain how the digoxigenin (DIG) DNA labeling and detection kit that

you used works? In other words, explain how DIG labeling and CSPD detection is done.

2. Explain how digoxigenin (DIG) DNA labeling and BCIP/NBT detection is similar and yet different from your answer to question 1.

3. Explain another way to label a DNA probe for Southern hybridization analysis?

4. Compare and contrast Southern blotting to northern blotting and microarray analysis.

5. What is the purpose of prehybridization?6. What two hybridization parameters or conditions are typically altered for

maximum hybridization?7. What are zoo blots and garden blots?8. What kinds of nucleic acid bind to nitrocellulose? What kinds of nucleic

acid do not bind?9. Why do you soak the gel and not the colony lift in 0.2N HCl?10. When doing a northern blot, why do you avoid soaking the gel in 0.5 M

NaOH/1.5 M NaCl as you do in Southern blotting?

What gene are you working with in this lab?

• The gene you cloned is At4g27960, which encodes the ubiquitin conjugating enzyme 9.

• The cloned gene fragment is 415 bp in size, and its GC content is 41%. This information is used for calculating the Tm.