13 October 1993
Press Release: The 1993 Nobel Prize in Chemistry
THE ROYAL SWEDISH ACADEMY OF SCIENCES
The Royal Swedish Academy of Sciences has
decided to award the 1993 Nobel Prize in Chemistry for contributions
to the development of methods within DNA-based chemistry,
with half to
Dr Kary B. Mullis, La Jolla, California, U.S.A., for his invention
of the polymerase chain reaction (PCR) method,
and half to
Professor Michael Smith, University of British Columbia, Vancouver,
Canada, for his fundamental contributions to the establishment of oligonucleotide-based,
site-directed mutagenesis and its development for protein studies.
Decisive progress in gene technology through two new methods: the polymerase
chain reaction (PCR) method and site-directed mutagenesis
The chemical methods that Kary B. Mullis and Michael Smith
have each developed for studying the DNA molecules of genetic material have
further hastened the rapid development of genetic engineering. The two methods
have greatly stimulated basic biochemical research and opened the way for
new applications in medicine and biotechnology.
The applications of Mullis' PCR method are already many. It is for
example possible using simple equipment to multiply a given DNA segment
from a complicated genetic material millions of times in a few hours, which
is of very great significance for biochemical and genetic research. The
method offers new possibilities particularly in medical diagnostics, and
is used, for example, for discovering HIV virus or faulty genes in hereditary
diseases. Researchers can also produce DNA from animals that became extinct
millions of years ago by using the PCR method on fossil material.
The genetic code programmed into the DNA molecule determines the number
and sequence of amino acids in a protein, and thus also the functional properties
of the protein. With Smith's method it is possible to re-programme
the genetic code and in this way replace specific amino acids in the proteins.
This is termed site-directed mutagenesis. The possibilities of studying
the structure and function of the protein molecules have changed fundamentally,
and hence also the possibilities of constructing proteins with new properties.
Attempts are being made, for example, to improve protein stability so that
proteins can manage technical processes, to tailor antibodies so that they
can attack cancer cells and to alter proteins to create faster-growing crop
strains. The term protein design has already become a concept.
Chemically, the genetic material of living organisms consists of DNA
(deoxyribonucleic acid). DNA molecules consist of two very long strands
twisted around each other to form a double helix. Each strand is formed
of smaller molecules, nucleotides, that represent the letters of the genetic
material. There are only four different letters, designated A, T, C and
G. The two DNA strands are complementary, being held together by A - T and
G - C bonds. It is only when the genetic code is to be read off e.g. for
protein building in the cell that the two strands are separated. The genetic
information in DNA exists as a long sentence of code words, each of which
consists of 3 letters which can be combined in many different ways (e.g.
CAG, ACT, GCC). Each three-letter code word can be translated by special
components within the cell into one of the twenty amino acids that build
up proteins. It is the proteins that are responsible for the functions of
living cells, including their ability to function, among other things, as
enzymes maintaining all the chemical reactions required for supporting life.
The proteins' three-dimensional structure and hence their function is determined
by the order in which the various amino acids are linked together during
The flow of genetic information goes from DNA via the translator molecule
RNA to the proteins. By re-programming the code of a DNA molecule, e.g.
changing the word CAC to GAC, it would be possible to obtain a protein in
which the amino acid histidine is replaced by the amino acid aspartic acid.
In nature, such mix-programming of the genetic material (mutation) occurs
randomly, and is nearly always fatal to the organism. However, a dream of
biochemical researchers has been to alter a given code word in a DNA molecule
so as to be able to study how the properties of the mutated protein differ
from the natural. It was through Smith's oligonucleotide-based site-directed
mutagenesis that this dream became reality. As early as the 1970s Smith
learned to synthesize oligonucleotides, short, single-strand DNA fragments,
chemically. He also studied how these synthetic fragments could bind a virus
to DNA. Smith then discovered that even if one of the letters of the synthetic
DNA fragment was incorrect it could still bind at the correct position in
the virus DNA and be used when new DNA was being synthesized. At the beginning
of the 1970s Smith was a visiting researcher at Cambridge and the story
goes that it was during a coffee-break discussion that the idea arose of
getting a reprogrammed synthetic oligonucleotide to bind to a DNA molecule
and then having it replicate in a suitable host organism. This would give
a mutation which in turn would be able to produce a modified protein. In
1978 Smith and his co-workers made this idea work in practice. They succeeded
both in inducing a mutation in a bacteriophagic virus and "curing"
a natural mutant of this virus so that it regained its natural properties.
Four years later Smith and his colleagues were able for the first time to
produce and isolate large quantities of a mutated enzyme in which a pre-determined
amino acid had been exchanged for another one.
A protein with a changed (mutated) amino acid can be produced with
site directed mutagenesis. A chemically synthesized DNA fragment with a
changed code word is bound to a virus DNA which is multiplied in a bacterium.
The DNA molecule with the changed code word is reduplicated and can be used
for producing the changed protein.
Smith's method has created entirely new means of studying in detail
how proteins function, what determines their three-dimensional structure
and how they interact with other molecules inside the cell. Site-directed
mutagenesis has without doubt revolutionised basic research and entirely
changed researchers' ways of performing their experiments. The method is
also important in biotechnology, where the concept protein design has been
introduced, meaning the construction of proteins with desirable properties.
It is already possible, for example, to improve the stability of an enzyme
which is an active component in detergents so that it can better resist
the chemicals and high temperatures of washing water. Attempts are being
made to produce biotechnically a mutated haemoglobin which may give us a
new means of replacing blood. By mutating proteins in the immune system,
researchers have come a long way towards constructing antibodies that can
neutralise cancer cells. The future also holds possibilities of gene therapy,
curing hereditary diseases by specifically correcting mutated code words
in the genetic material. Site-directed mutagenesis of plant proteins is
opening up the possibility of producing crops that can make more efficient
use of atmospheric carbon dioxide during photosynthesis.
The "Polymerase Chain Reaction" (PCR)
The PCR technique was first presented as recently as 1985 but is nevertheless
already one of the most widespread methods of analysing DNA. With PCR it
is possible to replicate several million times, in a test tube, an individual
DNA segment of a complicated genetic material. Mullis has described how
he got the idea for the PCR during a night drive in the Californian mountains.
Two short oligonucleotides are synthesized so that they are bound correctly
to opposite strands of the DNA segment it is wished to replicate. At the
points of contact an added enzyme (DNA polymerase) can start to read off
the genetic code and link code words through which two new double strands
of DNA are formed. The sample is then heated, which makes the strands separate
so that they can be read off again. The procedure is then repeated time
after time, doubling at each step the number of copies of the desired DNA
segment. Through such repetitive cycles it is possible to obtain millions
of copies of the desired DNA segment within a few hours. The procedure is
very simple, requiring in theory only a test tube and some heat sources,
even though there are now commercial PCR apparatuses that manage the whole
procedure automatically and with great precision.
The PCR method can be used for reduplicating a segment of a DNA molecule,
e.g. from a blood sample. The procedure is repeated 20-60 times, which can
give millions of DNA copies in a few hours.
As has site-directed mutagenesis, the PCR method has decisively improved
the outlook for basic research. The sequencing and cloning of genes has
been appreciably simplified. PCR has also made Smith's method of site-directed
mutagenesis more efficient. Since it is possible with PCR to perform analyses
on extremely small amounts of material, it is easy to determine genetic
and evolutionary connections between different species. It is very probable
that PCR combined with DNA sequencing is going to represent a revolutionary
new instrument for studies of the systematics of plant and animal species.
The biomedical applications of the PCR method are already legion. Now that
it is possible to discover very small amounts of foreign DNA in an organism,
viral and bacterial infections can be diagnosed without the time-consuming
culture of microorganisms from patient samples. PCR is now being used, for
example, to discover HIV infections. The method can also be exploited to
localise the genetic alterations underlying hereditary diseases. Thus PCR,
like site-directed mutagenesis, has a great potential within gene therapy.
Without the PCR method, the HUGO project, with its objective of determining
every single DNA code in, among other things, the human genetic material,
would hardly be realistic. In police investigations PCR can give decisive
information since it is now possible to analyse the DNA in a single drop
of blood or in a hair found at the scene of a crime.
Another fantastic application is that it is possible to mass-produce DNA
from fossil remains. Researchers have, for example, succeeded in producing
genetic material from insects that have been extinct for more than 20 million
years by using the PCR method on DNA extracted from amber. This possibility
has already inspired authors of science fiction. The very popular film "Jurassic
Park" is about the fear that arises when researchers using PCR recreate
extinct giant reptiles.
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