DNA Manipulation

The Biotechnology Era: Manipulating Genes and Designing Life

By the late 1960's and early 1970's, the mechanisms of DNA were understood well enough for some geneticists to begin proposing ways of changing and manipulating the genetics of an organism. Although selective breeding techniques had been used by farmers for centuries, and Mendelian (or classical) genetics had been used as a tool in the genetics lab for almost 60 years, the advent of technologies that could manipulate an actual DNA sequence, and even read the individual nucleotides of that sequence ushered in a new era of knowledge and possibility. These new technologies led to a new kind of geneticist; the molecular biologist. The first wave of technologies resulting from understanding intact biological systems, which were in turn used to manipulate sequences of DNA. The second wave separated biological molecules and processes from their organic settings, using them as tools in artificial environments such as test tubes and recombinant organisms.

Gel Electrophoresis

GelIn order to work with DNA molecules, it was necessary to be able to move sequences of DNA around, and separate particular sequences from each other. The electrophoretic method for separating such sequences was and is one of the most useful tools for this task. Gel electrophoresis was adopted from biochemistry. The basic process was developed by Arne Tiselius, who worked with proteins. In the late 1950's the first three-dimensional gels were attempted; these were made with starch. In the 1970's, Sequencing gels and agarose gels were developed and electrophoresis became closely linked with the visualization of DNA. Gel electrophoresis allowed geneticists and molecular biologists to isolate and separate specific DNA sequences.


Restriction Digest

Restriction EnzymesAs methods for visualizing DNA were being developed in the the1950's, a new tool was discovered; the restriction enzyme. The same physicist-biologist all-star teams that first described the structure and function of DNA (the phage group) were involved in reporting "endonuclease activity" of bacteria to fight infection by phages. Werner Arber proposed that an endonuclease cut the DNA of the phage by recognizing a specific sequence of A's, T's, G's, and C's in the phage DNA. He identified the first restriction enzyme in 1968, but it did not cut in the specific manner he described.

Using Arber's model, Hamilton Smith isolated the first restriction enzyme that cut at a specific DNA sequence in 1970. Soon many more were identified; they all cut DNA at specific sequences. In 1971 Daniel Nathans then began to use restriction enzymes to analyze longer and longer pieces of DNA. His work used restriction enzymes(which were from the realm of chemistry, and microscopic processes) in the context of genetics (which was based at that time on macroscopic organisms and whole populations), in order to identify distinct sequences of DNA and to create genetic maps.


Ligation

In 1966 B Weiss & CC Richardson discover DNA ligase, an enzyme that could be used to "ligate" or paste together two strands of DNA. In 1972, Paul Berg used a restriction enzyme to cut DNA, and then a ligase to paste two DNA strands together to form a hybrid circular molecule; this was the first recombinant DNA molecule. This method of using both restriction enzymes and ligase ushered in a new era of genetic manipulation.

What happened next outlines how funding basic scientific research can lead to results that the most focused and targeted commercial efforts cannot achieve, but that ultimately end up revolutionizing an industry. Looking back at the history of biotechnology now, it is easy to see the importance of certain projects. Nevertheless, at the time those experiments were being done and before the results were clear, no one could have predicted their impact on molecular biology, genetics, and indeed many aspects of our lives today.

Bacterial transformation and cloning: Genetic Engineering

In 1970, M Mandel & A Higa transformed bacteria with an existing gene, using a CaCl2 method. By1973, Stanley Cohen and Herbert Boyer had developed biochemical and genetic techniques to cut and paste DNA from different sources, and then transform that new "recombinant" DNA into bacteria. The recombinant DNA was then expressed in the bacteria and resulted in the first recombinant organism. This process, termed "cloning," defines the beginning of the biotechnology era. By 1976, the first working synthetic gene had been developed. That same year, Boyer and Robert Swanson founded Genentech, Inc., the first company to commercialize recombinant technologies. In 1977, Genentech, Inc., produced the first human protein manufactured in a bacteria.

DNA sequencing

The advent of molecular techniques for manipulating and editing sequences of DNA necessitated a need for a way to determine the correct order the As, Ts, Gs, and Cs that make up a unique sequence of DNA. Molecular biologists needed some kind of translator for their toolbox in order to read the specific sequences of the genes they were working with. In 1975, Frederick Sanger and Alan Coulson developed the first method for sequencing DNA. Two years later, Walter Gilbert and Allan Maxam devised a method for sequencing DNA using chemicals rather than enzymes.

PCR/DNA Amplification

PCRBy the mid-1980's the biotechnology revolution was in fun effect. The was a blossoming industry of companies attempting to create both medicines and tools for molecular biology, Moreover, researchers in both academic and commercial labs were creating new ideas and methods for working with DNA. The most important innovation from this period was the polymerase chain reaction (PCR), which was a method for DNA amplification.

Kary MullinsIn 1985, while behind the wheel of his car, Kary Mullis had an eureka moment. Mullis took existing processes and linked them together is such a way that the result ended up revolutionizing many aspects of molecular biology. One of the most important areas was in sequencing DNA.

In 1986, Caltech and Applied Biosystems, Inc., created an automated DNA fluorescence sequencer, which was based on the PCR principle. Three years later, the National Center for Human Genome Research was created to map and sequence all human DNA. In 1990, this effort was refocused as The Human Genome Project, an international effort to map all of the genes in the human body. Sequencing technology developments moved this effort almost 7 years ahead of schedule, and in 1998 a rough draft of the human genome map was produced.