Basic techniques - Principles of Gene Manipulation - page 11

electrophoretic mobility. The assay typically involves
the addition of protein to linear double-stranded DNA
fragments, separation of complex and naked DNA
by gel electrophoresis and visualization. A review of
the physical basis of electrophoretic mobility shifts and
their application is provided by Lane et al . (1992).

• blotting of nucleic acids from gels;
• dot and slot blotting;
• colony and plaque blotting.
Colony and plaque blotting are described in detail on
pp. 104 -105 and dot and slot blotting in Chapter 14.

Southern blotting

Nucleic acid blotting

The original method of blotting was developed by
Southern (1975, 1979b) for detecting fragments in
an agarose gel that are complementary to a given
RNA or DNA sequence. In this procedure, referred to
as Southern blotting, the agarose gel is mounted on
a filter-paper wick which dips into a reservoir con-
taining transfer buffer (Fig. 2.5). The hybridization
membrane is sandwiched between the gel and a
stack of paper towels (or other absorbent material),
which serves to draw the transfer buffer through the
gel by capillary action. The DNA molecules are car-
ried out of the gel by the buffer flow and immobilized
on the membrane. Initially, the membrane material
used was nitrocellulose. The main drawback with
this membrane is its fragile nature. Supported nylon
membranes have since been developed which have
greater binding capacity for nucleic acids in addition
to high tensile strength.
For efficient Southern blotting, gel pretreatment is
important. Large DNA fragments (

Nucleic acid labelling and hybridization on mem-
branes have formed the basis for a range of experi-
mental techniques central to recent advances in our
understanding of the organization and expression
of the genetic material. These techniques may be
applied in the isolation and quantification of specific
nucleic acid sequences and in the study of their
organization, intracellular localization, expression
and regulation. A variety of specific applications
includes the diagnosis of infectious and inherited
disease. Each of these topics is covered in depth in
subsequent chapters.
An overview of the steps involved in nucleic acid
blotting and membrane hybridization procedures is
shown in Fig. 2.4. Blotting describes the immobiliza-
tion of sample nucleic acids on to a solid support,
generally nylon or nitrocellulose membranes. The
blotted nucleic acids are then used as 'targets' in
subsequent hybridization experiments. The main
blotting procedures are:

10 kb) require a
longer transfer time than short fragments. To allow

>

Immobilization of nucleic acids

• Southern blot
• Northern blot
• Dot blot
• Colony/plaque lift

Pre-hybridization

Labelled DNA
or RNA probe

Hybridization Removal of probe

prior to reprobing

Stringency washes

Fig. 2.4 Overview of nucleic acid
blotting and hybridization (reproduced
courtesy of Amersham Pharmacia
Biotech).

Detection