Biology (Holt)

SECTION 1Genetic Engineering 231

The word electrophoresisis

from the Latin electrocus,

meaning “electricity,” and

the Greek phoresis,mean-

ing “to carry.” Knowing this

makes it easier to remember

that electrophoresis uses

electricity to separate

DNA fragments.

BIO

graphic

DNA from each bacterial

clone colony is cut with

restriction enzymes.

A DNA or RNA probe can be used to identify a cloned gene.

Southern Blot: Identifying a Gene of Interest

1 DNA fragments are separated by gel

electrophoresis.

2 DNA is transferred to filter paper (blotted).

A probe is added.

3 Only DNA frag-ments that contain

the gene of interest

bind to probes.

Colony I

has the

gene of

interest.

4

Probe

III

III III III

Gel

+

Filter paper

Figure 5

Confirmation of a Cloned Gene

The surviving bacterial colonies are tested for the presence of the
gene of interest. One method used to identify a specific gene is a
technique called a Southern blot, as summarized in Figure 5.

Step In a Southern blot, the DNA from each bacterial clone colony

is isolated and cut into fragments by restriction enzymes.

Step The DNA fragments are separated by gel

(ee LEKtroh fuh REE sis), a technique that uses an electric

field within a gel to separate molecules by their size. The

gel is a rectangular slab of gelatin with a line of little rec-

tangular wells near the top edge. The DNA sample is placed

in the pits. Because DNA is negatively charged, it migrates

toward the positive pole when the electric field is applied.

The DNA fragments move through the gel, with the small-

est DNA fragments moving fastest. A pattern of bands is

formed. The gel is soaked in a chemical solution that sepa-

rates the double strands in each DNA fragment into single-

stranded DNA fragments.

Step The DNA bands are then transferred (blotted) directly onto

a piece of filter paper. The filter paper is moistened with

a probe solution. are radioactive- or fluorescent-

labeled RNA or single-stranded DNA pieces that are

complementary to the gene of interest.

Step Only the DNA fragments complementary to the probe will

bind with the probe and form visible bands.

Probes

electrophoresis