Nucleic Acid

Hybridization, Probes,
and Blotting Techniques
DM Bascos
MBB 141
Nucleic acid hybridization

Technique used to detect DNA or RNA of a
particular sequence

DNA or RNA is immobilized on a membrane

Uses probes specific for DNA or RNA segment of
interest

Design

Optimization of hybridization conditions
Southern blot

Developed by
Professor Sir Edwin
Southern

Used to detect DNA

'You don't just develop
things out of the blue',
he says. 'Questions
trigger thinking about
how to solve them.'
Northern blot

Developed by James Alwine and David Kemp

For detecting RNA
Basic process of blotting
Purify DNA

Digest with RE Purify RNA

Run on agarose gel

Transfer to membrane
How to transfer nucleic acids

Weight

Glass plate

Paper towels

Filter paper
Membrane
Agarose gel

“Wick”

Tray with transfer buffer

The transfer process

By capillary action

Done in a few hours (sometimes overnight)

Must make sure there are NO BUBBLES
between layers

Why?

How do we do this?
Things to remember

NEVER TOUCH the membrane with bare
hands!!!

Gel must be “reversed”
Things to consider

Longer DNA/RNA transfers slower than short
DNA/RNA

DNA and RNA must be single stranded for
hybridization to occur

How do we deal with these?

must denature and nick nucleic acids
Pre-treating the nucleic acids

For DNA

short depurination treatment with 0.25 M HCl

follow with alkali

must equilibriate gel in neutral buffer before
transfer

For RNA

denature RNA with formaldehyde
Membranes

Serves as solid support for DNA

Types

nitrocellulose

nylon

Cross-linking of DNA

heat

UV
Probes

single stranded DNA or RNA

complementary to target sequence

labeled

radioactive – 32P

non-radioactive – enzyme (e.g. DIG, AP, HRP)
Probe design

Must be specific for target sequence

The longer, the better (in general)

why?

GC content

Degree of mismatch
Hybridization protocol
Pre-soak blot in hybridization solution

Denature probe
(i.e., boil)

Add probe

Incubate 5 hours or more

Lots of washes
(increasing temp &/or salt concentration)

Detect
Stringency

Reduce non-specific binding

Factors to consider

Probe

sequence

length

Temperature of hybridization

Tm = 81.5oC + 16.6logM + 0.41(%G+C)

Salt concentration for washes

Temperature for washes

Tm = 4 x (number of GC pairs) + 2 x (number of AT pairs)
Detection

For radioactive probes

Autoradiography

Expose blot to x-ray film and develop

For non-radioactive probes

e.g. DIG

Add anti-DIG antibody with Alkaline Phosphatase

Add AP substrate

produce precipitate on blot
Autoradiography
image from Roche’s Introduction to DIG system and DIG application manual
Sample

image from http://www.mun.ca/biology/scarr/Southern_Blot_analysis.html

Alternative blotting techniques

Electrophoretic transfer

Vacuum transfer
Other blotting techniques

Dot and slot blots

Colony and plaque blotting
Dot or slot blot
Colony or plaque blot

image from
http://www.ebiotrade.com/BUYF/productsf/qiagen/QIAexpress%20Detectio
n%20Systems.htm