DETECTION AND ANALYSIS OF NUCLEIC ACIDS - A Guide to Methods in the Biomedical Sciences - page 27

DNA from one bacterium to another (and its daughter cells). As is often
true of ground-breaking discoveries, the acceptance of this one was tem-
pered by skepticism due to the prevailing wisdom of the time that proteins
were responsible for heredity. Indeed, the composition of the nucleic
acids that made up the DNA was considered to be too simple to be re-
sponsible for something as complex as a gene. However, the conclusion
from Avery's research was unmistakable: nucleic acids, and thus DNA,
was responsible for the genetic makeup of an organism. How DNA was
responsible for the transmission of genetic traits could only be under-
stood after Watson and Crick had solved the 3-dimensional structure of
DNA and the genetic code had been broken. But these ground-breaking
studies would not have been possible without the work of Avery. While
the awarding of a Nobel Prize is sometimes controversial (we'll come
back to that later), the failure to award one to Avery has been considered
by some to be one of the most egregious oversights in Nobel history.
Within 20 years of the discovery of the 3-dimensional structure of
DNA, its chemical structure was solved, DNA replication was under-
stood, and the basic parameters of how DNA is precisely transcribed
into RNA (ribonucleic acid), which is then translated into protein, be-
came clear. The development of many new technologies was required
to achieve these breakthroughs, and many of the techniques that were
invented for these discoveries remain in use today. Some of these were
so instrumental in their applicability to solve biological problems that
they also garnered Nobel Prizes for their inventors (Table 2).
The following are brief descriptions of some of the common methods
used to manipulate nucleic acids, especially DNA.

B. Basic methods for nucleic acid analysis

Gels and gradients for separation of nucleic acids

The size of a piece of DNA, usually produced as the result of restriction
digestion (see below) can be determined by separation on gradients or
gels. For gradient separation, sucrose density gradients were common
at one time but were supplanted by gels once electrophoretic separation
techniques were developed. Two types of gel matrices are commonly
used for the separation of nucleic acids: those made from polyacry-
lamide, and those using agarose. When a mixture of DNA fragments
is applied to one end of the gel and subjected to an electric current,
the DNA fragments migrate toward the positive pole of the field, the an-
ode, due to the negatively charged phosphate groups that make up the
DNA backbone. Following electrophoretic separation, the DNA can be
visualized by one or more methods as discussed below.