19 December 2012

Synthetic DNA



The invention: 

A method for replicating viral deoxyribonucleic
acid (DNA) in a test tube that paved the way for genetic engineering.

The people behind the invention:

Arthur Kornberg (1918- ), an American physician and
biochemist
Robert L. Sinsheimer (1920- ), an American biophysicist
Mehran Goulian (1929- ), a physician and biochemist




The Role of DNA

Until the mid-1940’s, it was believed that proteins were the
carriers of genetic information, the source of heredity. Proteins
appeared to be the only biological molecules that had the complexity
necessary to encode the enormous amount of genetic information
required to reproduce even the simplest organism.
Nevertheless, proteins could not be shown to have genetic properties,
and by 1944, it was demonstrated conclusively that deoxyribonucleic
acid (DNA) was the material that transmitted hereditary
information. It was discovered that DNA isolated from a
strain of infective bacteria that can cause pneumonia was able to
transform a strain of noninfective bacteria into an infective strain;
in addition, the infectivity trait was transmitted to future generations.
Subsequently, it was established that DNA is the genetic material
in virtually all forms of life.
Once DNA was known to be the transmitter of genetic information,
scientists sought to discover how it performs its role. DNA is a
polymeric molecule composed of four different units, called “deoxynucleotides.”
The units consist of a sugar, a phosphate group, and a
base; they differ only in the nature of the base, which is always one of
four related compounds: adenine, guanine, cytosine, or thymine. The
way in which such a polymer could transmit genetic information,
however, was difficult to discern. In 1953, biophysicists James D.Watson
and Francis Crick brilliantly determined the three-dimensional
structure of DNAby analyzing X-ray diffraction photographs of DNA
fibers. From their analysis of the structure of DNA,Watson and Crick
inferredDNA’s mechanism of replication. Their work led to an understanding
of gene function in molecular terms.
Watson and Crick showed that DNA has a very long doublestranded
(duplex) helical structure. DNAhas a duplex structure because
each base forms a link to a specific base on the opposite
strand. The discovery of this complementary pairing of bases provided
a model to explain the two essential functions of a hereditary
molecule: It must preserve the genetic code from one generation to
the next, and it must direct the development of the cell.
Watson and Crick also proposed that DNA is able to serve as a
mold (or template) for its own reproduction because the two strands
ofDNApolymer can separate. Upon separation, each strand acts as a
template for the formation of a new complementary strand. An adenine
base in the existing strand gives rise to cytosine, and so on. In
this manner, a new double-stranded DNAis generated that is identical
to the parent DNA.


DNA in a Test Tube

Watson and Crick’s theory provided a valuable model for the reproduction
of DNA, but it did not explain the biological mechanism
by which the process occurs. The biochemical pathway of DNA reproduction
and the role of the enzymes required for catalyzing the
reproduction process were discovered by Arthur Kornberg and his
coworkers. For his success in achievingDNAsynthesis in a test tube
and for discovering and isolating an enzyme—DNA polymerase—
that catalyzed DNA synthesis, Kornberg won the 1959 Nobel Prize
in Physiology or Medicine.
To achieve DNAreplication in a test tube, Kornberg found that a
small amount of preformed DNA must be present, in addition to
DNApolymerase enzyme and all four of the deoxynucleotides that
occur in DNA. Kornberg discovered that the base composition of
the newly made DNAwas determined solely by the base composition
of the preformed DNA, which had been used as a template in
the test-tube synthesis. This result showed that DNA polymerase
obeys instructions dictated by the template DNA. It is thus said to
be “template-directed.” DNA polymerase was the first templatedirected
enzyme to be discovered.
Although test-tube synthesis was a most significant achievement,
important questions about the precise character of the newly
made DNAwere still unanswered. Methods of analyzing the order,
or sequence, of the bases in DNA were not available, and hence it
could not be shown directly whetherDNAmade in the test tube was
an exact copy of the template of DNA or merely an approximate
copy. In addition, some DNAs prepared by DNA polymerase appeared
to be branched structures. Since chromosomes in living cells
contain long, linear, unbranched strands of DNA, this branching
might have indicated that DNA synthesized in a test tube was not
equivalent to DNA synthesized in the living cell.
Kornberg realized that the best way to demonstrate that newly
synthesizedDNAis an exact copy of the original was to test the new
DNAfor biological activity in a suitable system. Kornberg reasoned
that a demonstration of infectivity in viral DNA produced in a test
tube would prove that polymerase-catalyzed synthesis was virtually
error-free and equivalent to natural, biological synthesis. The
experiment, carried out by Kornberg, Mehran Goulian at Stanford
University, and Robert L. Sinsheimer at the California Institute of
Technology, was a complete success. The viral DNAs produced in a
test tube by the DNA polymerase enzyme, using a viral DNA template,
were fully infective. This synthesis showed that DNA polymerase
could copy not merely a single gene but also an entire chromosome
of a small virus without error.


Consequences





The purification of DNApolymerase and the preparation of biologically
active DNA were major achievements that influenced
biological research on DNA for decades. Kornberg’s methodology
proved to be invaluable in the discovery of other enzymes that synthesize
DNA. These enzymes have been isolated from Escherichia
coli bacteria and fromother bacteria, viruses, and higher organisms.
The test-tube preparation of viral DNA also had significance in
the studies of genes and chromosomes. In the mid-1960’s, it had not
been established that a chromosome contains a continuous strand of
DNA. Kornberg and Sinsheimer’s synthesis of a viral chromosome
proved that it was, indeed, a very long strand of uninterrupted
DNA.
Kornberg and Sinsheimer’s work laid the foundation for subsequent
recombinant DNAresearch and for genetic engineering technology.
This technology promises to revolutionize both medicine
and agriculture. The enhancement of food production and the generation
of new drugs and therapies are only a few of the subsequent
benefits that may be expected.


See also : Artificial hormone; Cloning; Genetic“fingerprinting”;
Genetically engineered insulin; In vitro plant culture;
Synthetic amino acid;Artificial gene synthesis.


Further Reading


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