In 1975, Edwin M. Southern developed procedures for detecting specific DNA
fragments so that a particular gene could be isolated from a complex DNA mix-
ture. This technique is called the Southern blotting technique. DNA fragments are
separated by size with agarose gel electrophoresis. Gel electrophoresis takes
advantage of the chemical and physical properties of DNA to separate the frag-
ments. The phosphate groups in the backbone of DNA are negatively charged.
This makes the DNA molecules attracted to anything that is positively charged.
In gel electrophoresis the DNA molecules are placed in an electric field so that
they migrate towards the positive charge.
The DNA is placed in agarose, a semi solid gelatin, and placed in a tank of
buffer. When electrical current is applied, the DNA molecules migrate through
the agarose gel, separate, and travel toward the positive poles of the electric
fields. The entire DNA fragments migrates through the gel. The larger DNA frag-
ments have a harder time moving than the smaller ones, so the small fragments
travel farther through the gel.
ARTIFICIAL DNA: PUT TING TOGETHER THE PIECES
Oligonucleotides, from the Greek word oligomeaning “few,” are short pieces
of DNA or RNA that are 2 to 30 nucleotides long. The ability to synthesize DNA
oligonucleotides of a known sequence is incredibly important and useful. A
DNA probe is used to analyze fragments of DNA. A DNA probe is a single-
stranded fragment of DNA that recognizes and binds to a complementary sec-
tion of DNA in a mixture of DNA molecules.
DNA probes can be synthesized and DNA fragments can be prepared for use
in molecular techniques such as polymerase chain reaction (PCR). Polymerase
chain reaction is a technique that was developed by Kary Mullis in 1985. It pro-
CHAPTER 8 Recombinant DNA Technology^133
Microbial Recognition Cleavage
Enzyme Source Sequence Sites (↓,↑) End Product
EcoRI Escherichia coli GAATTC G↓AATTC G AATTC
CTTAAG CTTAA↑G CTTAA GTable 8-1. Recombinant DNA Is DNA with a New Sequence Formed by Joining Recognition
Sequence Fragments from Different Sources