Discovery of PCR
Inventor of PCR
PCR, or Polymerase Chain Reaction was first popularly thought to have been conceived by Dr. Kerry Mullis in 1983 while working at the Cetus Corporation in Emeryville, CA, along with other researchers at Cetus Corporation. Cetus Corporation discovered a method to start and stop DNA polymerase enzyme activity at specific points along a single strand of DNA. On the other hand, some pioneering research was also done by Gobind Khorana, who described a basic principle of replicating a piece of DNA using two primers (1971). Kerry Mullis discovered that by harnessing this component of molecular reproduction technology, a target DNA of interest could be amplified exponentially. This DNA amplification procedure was an in vitro process (meaning in a test-tube).
Cell-free DNA amplification by PCR was able to simplify many of the standard procedures for DNA cloning, DNA analysis, and the modification of DNA. Previous molecular biology techniques for isolating a specific piece of DNA had relied on gene cloning, which is a tedious and slow procedure. PCR, as Kerry Mullis stated “lets you pick the piece of DNA you’re interested in and have as much of it as you want”.
Progress for the development was initially limited by primer synthesis and polymerase purification issues. When Cetus scientists eventually succeeded in making the polymerase chain reaction perform as desired in a reliable fashion, they had an immensely powerful technique for providing almost unlimited quantities of the precise genetic material molecular biologists and others required for their research. Since the first report in1985, more than 5000 scientific papers were published by 1992.
Kerry Mullis's PCR Idea
In Mullis’s head, the discovery grew from a theoretical scheme to perform limited dideoxynucleotide sequencing of unique human genes using synthetic oligonucleotides for the purpose of diagnosing common human disease mutations. An obvious obstacle to such a direct sequencing strategy was the high complexity of the human genome (3.3 X 10^9 base pairs). Therefore, a second oligonucleotide or primer was added to block the progression of the synthesis of the first primer. Later in his thinking however, the second primer was included to bind to the other DNA strand, so that each strand of the mutant allele would contribute to the eventual signal. If the scheme involving simultaneous hybridization of primers to each strand was changed by heating the mixture and then repeating the annealing and extension steps, then the primary signal would be increased even more. Repetition of the steps would allow the products of the first cycle to be duplicated in the second cycle, to yield two copies. Repeating the steps in the cycle again would result in four copies, et cetera.
Several weeks passed before this great idea was even attempted at the company labs. Eventually, two oligonucleotide primers were synthesized to be perfectly complementary to each end of the 110 base pair region of a cloned segment of the human beta-globin gene, the amplification was performed, and the DNA products were identified by acrylamide gel electrophoresis. The end result was the anticipated 110 base pair DNA fragment, and the beginning of PCR as a basic technique in molecular biology.
Initial Problems With PCR
In Mullis's original PCR process, the enzyme was used in vitro. The double-stranded DNA was separated into two single strands of DNA by heating it to 96°C. At this temperature, however, the E.Coli DNA polymerase was destroyed, so that the enzyme had to be replenished with new fresh enzyme after the heating stage of each cycle. Mullis's original PCR process was very inefficient since it required a great deal of time, vast amounts of DNA-Polymerase, and continual attention throughout the PCR process. Other researchers and companies had to greatly improve the technology and enzymes with the result that PCR now is home to hundreds if not thousands of companies and research institutes which work on improving PCR quality, fidelity, and the processivity of PCR (the length of DNA that can be amplified).
See PCR Development and History