Long before PCR achieved mainstream recognition, however, LBNL researchers used PCR to help map and sequence the three billion letters of the human genetic code.
Now, engineers in LBNL's Human Genome Center instrumentation group, working closely with Lab biologists, have developed an improved type of PCR apparatus that can perform the required steps in less than half the time. Chris Martin of the HGC's sequencing group says the next, even faster, version of the new machine, now under development, could lead to a total robotic automation of the process.
PCR involves three steps (see article, "How PCR Works" at bottom of this file), each of which must be performed at a specific temperature. To be most effective, the temperature changes should be as rapid as possible. In conventional PCR equipment, an array of tubes or vials holding samples of DNA is placed in a metal block, and the temperature of the samples is controlled by heating and cooling the block.
In the new apparatus, known as the rapid thermal cycler, the temperature is controlled with circulating water, which results in faster temperature changes, more samples processed per hour, and an improved signal-to-noise ratio in the data. The new design reduces sample preparation and handling procedures by about 40 minutes per array.
In each step of the PCR process, precise regulation and rapid switching of temperature is crucial. In the prototype, designed by Tony Hansen, the DNA samples are held in a standard plastic microtiter plate half-submerged in water fed from three separate tanks, maintained at 95, 72, and 55 degrees centigrade. The user selects one of several preprogrammed timing protocols, and a system of computer-controlled valves switches the water for each step. This model has been operating for about 18 months in the HGC laboratories in Bldg. 74.
An improved version of the rapid thermal cycler is in the final stages of development. In this model, designed by Kanchi Karunaratni, the heating tanks, interlocks, and valves are all directed by a sophisticated process controller (similar to the computers that run automated factory assembly lines). This results in more precise regulation of temperature and more flexible switching of the valves. The new model is also smaller and more energy efficient. Three models of this version are being built, with the first due to go on line at HGC this summer.
In addition to Hansen and Karunaratni, LBNL engineers and technicians involved in the design of the rapid thermal cycler include Dave Wilson, Davey Hudson, Charlie Reiter, and Don Uber (software). Joe Jaklevic heads the HGC instrumentation group.
In the first step, denaturing, the test tube is heated close to boiling for a few seconds. This causes the double-stranded DNA to separate into two single strands. The primers bind to the exposed single strands at places where the sequence of primer bases is complementary to that of the DNA.
The second step is annealing. The temperature of the test tube is lowered to about 55 degrees centigrade for a few seconds, causing the primers to bind permanently to their sites on the single-stranded DNA. The DNA of interest is now single-stranded along most of its length, with a few small double-stranded areas where primers have aligned themselves.
The third step is extending. The temperature is raised to about 72 degrees centigrade for about a minute, which causes the polymerase protein to go to work. It moves along the single-stranded portion of the DNA, beginning at a primer, and creates a second strand of new DNA to match the first. After extension, the DNA of interest is double-stranded again, and the number of strands bearing the sequence of interest has been doubled.
The three steps are repeated about 30 times, resulting in an exponential increase of up to a billion-fold of the DNA of interest. A fragment of DNA that accounted for one part in three million in the original sample now fills the whole test tube.