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