Southern and Northern blotting

Comparison of Southern, Northern,

and Western analyses of Gene X

SOUTHERN BLOTTING

The technique was developed by E.M. Southern in 1975.

The Southern blot is used to detect the presence of a particular piece of DNA in a sample.

The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome.

Southern hybridization

Transfer buffer

Flow chart of Southern hybridization Preparing the samples and running the gel

Southern transfer

Probe preparation

Prehybridization

Hybridization

Post-hybridization washing

Signal detection

Isotope

Non-isotope

1) DNA Fragmentation

The DNA sample is digested with one or more restriction enzymes.

Nuclease hydrolyze the bonds connect bases within the strand.

If large amount of restriction fragments

faint smears rather than discrete bands

Same or nearly the same length

migrate together during electrophoresis

2) Gel Electrophoresis

Separate the fragments according to size to be detected using ethidium bromide.

Smaller fragments are migrating faster than larger fragments.

Gel Treatment

Double stranded DNA denatured using NaOH (base). separating it into single DNA strands for later hybridization to the single stranded probe.

If too large, treated with HCL (acid) to depurinate & break it into smaller fragment & neutralized before proceed.

Gel treatment

Acid treatment

• 0.2 N HCl solution

Denaturation

• NaOH solution

Neutralization

• Tris-Cl buffer (pH8.0)

Depurination

Optional

Occurs before neutralization

When DNA fragments is > 15 kilobases, it is

too hard to be transferred to filter

The gel is treated with dilute acid (0.2 M HCl

for 15 minutes)

• depurinate DNA fragment into smaller pieces

and promote higher efficiency transfer to filter.

Neutralization

DNA is placed into an alkaline solution containing

0.5mM NaOH to denature the dsDNA into ssDNA and

neutralize the acid in previous step.

Function:

(1) improve binding of the –vely charged DNA to

+vely charged filter

(2) ssDNA strands for hybridization

(3) destroy any remaining RNA present in the sample

Only ssDNA can be

transferred to filter

3. Blotting

Exert pressure

evenly to a gel to

ensure even contact

between gel and

membrane

Transfer is done by

capillary action which

draws buffer (binds ssDNA)

up onto the membrane

The binding of DNA

to membrane is due

to ion exchange

interactions

Southern transfer

Measure gel and set up transfer

assembly:

• Wick in tray with 20x SSC

• Gel

• Nitrocellulose or Nylon filters

(soaked in H2O and 20x SSC)

• 3MM Whatman filter paper

• Paper towels

• Weight

Blotting

Transferred the gel onto a membrane which is a sheet of nitrocellulose filter or nylon membrane to carry out blotting.

Enable the DNA fragments accessible by the label probes required for analysis by autoradiography or other detection methods depend on your probes.

Blotting

Gel is placed on the filter paper dipped in a reservoir containing transfer buffer. The membrane is laid on top of the gel wit paper towels placing on top of the membrane. Buffer lift up by capillary action to move the DNA from the reservoir to the membrane.

Blot permanently

Binding of the DNA to the membrane is weak after blotting (unstable).

Easily to washed off during washing.

A) Treated the DNA fragments with UV light

B) Oven baked it around 80°C.

4) Pre Hybridization- Blocking

To block non specific sites.

To ensure the specificity of the binding of the probe to the sample DNA.

5) Probe Labeling

The probe DNA is labeled so that it can be detected.

A)Radioactive labeling

B)Tagging with non radioactive probes

6) Hybridization

Process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA. The reactions are specific where the probes will only bind to targets with a complementary sequence. The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.

7) Post Hybridization- Washing

Excess unbound probe is washed away with buffers containing NaCl and detergent.

8) Detection

Labeled probe bound to the membrane is detected by autoradiography. Reveals the position of DNA fragment to which the probe hybridized. Autoradiograph is an image on an x-ray film which will visualized the discrete bands directly caused by beta emissions from the 32P radioactivelabeled probe. Hybridization of the probe to the sample DNA fragment on the membrane indicates that the fragment contains DNA sequence that is complementary to the probe.

Autoradiography

Exposure to x-ray

film

SOUTHERN BLOTTING

The key to this method is hybridization.

Hybridization-process of forming a

double-stranded DNA molecule between

a single-stranded DNA probe and a

single-stranded target patient DNA.

Step 3. DNA Denaturation

T G A A T C

A C A T T G

• Eliminate hydrogen bonds with sodium

hydroxide (NaOH)

Step 4. Transfer DNA to Membrane

• Two methods for transferring DNA to a

membrane

– capillary

– electrophoretic

Other Applications

DNA fingerprinting

• RFLP of VNTRs

Dot or slot blot

Colony or plaque lifts

Microarray analysis

Other Applications

DNA fingerprinting

• RFLP of VNTRs

Dot or slot blot

Colony or plaque lifts

Microarray analysis

Other Applications

DNA fingerprinting

• RFLP of VNTRs

Dot or slot blot

Colony or plaque lifts

Microarray analysis

Other Applications

DNA fingerprinting

• RFLP of VNTRs

Dot or slot blot

Colony or plaque lifts

Gene Expression

Other Applications

Microarray technology

Northern Blotting

Almost the same with Southern blot. Northern blotting is used for detecting RNA fragments. Southern blotting is used for detecting DNA fragments. Technique for detecting specific RNA separate by electrophoresis using RNA probe.

Northern blotting

Technique for detecting specific RNAs

separated by electrophoresis by

hybridization to a labeled DNA probe.

Application

Advantages

Northern blotting is able

to detect small changes in

gene expression that

microarrays cannot.

Disadvantages

Thousand of genes

visualized at a time with

microarray, while northern

blotting is usually looking at

one or small number of

genes

The flow chart of Northern hybridization

Prepare RNA samples and run RNA gel

Northern transfer

Probe preparation

Prehybridization

Hybridization

Post-hybridization washing

Signal detection

Isotope

Non-isotope

An example of Northern blotting

Northern blot

RNA gel 28 S

18 S

Comparison of

Southern and Northern Blot

Southern blotting Northern blotting

Molecule detected

DNA mRNA

Gel electrophoresis

Agarose gel Formaldehyde agarose gel

Gel pretreatment

Depurination, denaturation, and

neutralization

-

Blotting method

Capillary transfer Capillary transfer

Probes DNA Radioactive or nonradioactive

cDNA, Radioactive or nonradioactive

Detection system

Autoradiography Chemiluminescent

Colorimetric

Autoradiography Chemiluminescent

Colorimetric