Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-06-23
2002-10-29
Sisson, B. L. (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C435S183000, C435S287200, C536S023100, C536S024330, C034S080000
Reexamination Certificate
active
06472186
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a high-speed process for amplifying DNA, and a companion apparatus for automating this process, based upon the Polymerase Chain Reaction (PCR). Specifically, the present invention relates to a process in which pressurized gas is used to heat and cool a biochemical reaction chamber. This novel process has been successfully automated, as demonstrated by its ability to amplify DNA from picogram to microgram quantities on an unprecedented time scale.
2. Significance
Anyone who has ever driven an automobile or flown in a jet airplane can attest to the speed and reliability of pressurized gas machines. Pressurized gas devices include internal combustion engines, jet turbines, rockets, pneumatic airtools, and gas chromatographs. However, gas machines are not widely used in biochemistry. Described herein is a novel process for high-speed Polymerase Chain Reaction amplification of DNA, which relies upon the use of a pressurized gas thermocycler with high speed valves.
Five features of this pressurized gas thermocycler are important: (1) It is the fastest automated PCR device ever built; 30 cycles of amplification of an 85 base pair (b.p.) DNA fragment were carried out in 78 seconds. (2) With proper engineering, it can be made even faster. (3) It is compatible with on-line, fluorescent dye-based DNA detection optics. (4) Unlike any device which has been previously described, the speed of DNA thermocycling is limited by the biochemistry rather than the dead time of the thermocycler. In high-speed gas phase PCR, the rate of Taq Polymerase elongation (~80 nucleotides/sec at 72° C.) is rate-limiting. Theoretically, if faster DNA Polymerases (>1000 nucleotides/sec) can be found which are compatible with high-speed gas phase PCR, then even faster thermocycling times (<10 sec/30 cycles) are possible. (5) As an added benefit, high-speed gas phase PCR is generally more accurate than slower methods, probably because false reaction products have so little time to anneal and/or elongate.
Pressurized gas thermocyclers should prove especially useful in the diagnosis of life-threatening diseases where speed is essential. The present invention has major implications for DNA-based diagnoses used in biomedical research, genetics, molecular medicine, agriculture, veterinary science, and forensics.
3. The Background Art
[a] The Polymerase Chain Reaction. In order to understand how and why pressurized gas thermocyclers were built, one must first understand the Polymerase Chain Reaction (PCR) and how it has previously been automated. The Polymerase Chain Reaction is one of the most widely used techniques in molecular biology (U.S. Pat. No. 4,683,202 to Mullis; Saiki et al., 1985; Erlich, 1989; Mullis et al., 1994). PCR-amplified DNA can be used to diagnose mutations responsible for human genetic diseases (Kogan et al., 1987), in blood and tissue typing (Saiki et al., 1989a), or to detect pathogens responsible for important infectious diseases (Persing et al., 1993).
In a typical PCR reaction, template DNA sequences lying between the ends of two defined oligonucleotide primers can be amplified in 1 to 2 hours. Three sequential steps are normally employed: (i) double-stranded DNA is denatured (D) to a single-stranded form at a high temperature (90° C. to 95° C.), (ii) the resulting single-stranded DNA strands are annealed (A) to oligonucleotide primers at ~40° C. to 60° C., and (iii) primer template complexes are elongated (E) using a thermostable DNA Polymerase such as
Thermus aquaticus
(Taq) Polymerase at ~72° C. (Saiki, 1989b).
One cycle of these three steps (denaturation/annealing/elongation) results in a two-fold amplification of a DNA fragment whose 5′ and 3′ ends are defined by sequence-specific annealing of the oligonucleotide primers to the DNA template. Therefore, 30 PCR cycles result in a 2
30
-fold (~10
6
-fold) amplification of a particular DNA sequence. DNA is thus amplified from picogram to microgram amounts, which can be detected by standard analytical methods, such as gel electrophoresis, DNA hybridization, or optically.
[b] Automated PCR Instruments. A variety of machines have been built which automate the three-step PCR amplification process (Oste, 1989; Oste, 1994; Newton, 1995; Johnson, 1998). Generally, these devices may be classified into two categories: robotic devices which move the DNA samples to the heat; and thermocyclers which bring the heat to the samples.
Robotic devices such as Stratagene's ROBOCYCLER move tubes containing PCR reaction samples to and from a series of heat baths, which are thermostated at different temperatures. Although these devices may be useful in certain research applications, they are incapable of high-speed PCR. They require>60 minutes for 30 cycles of amplification.
Since the late 1980s, thermocyclers have become familiar devices in many biochemistry laboratories. Most commercially available PCR devices (Perkin-Elmer, MJ Research, Ericomp, Techne, Eppendorf, BioRad, Hybaid) are thermocyclers (Johnson, 1998). In general, two types of thermocyclers are employed: programmable heat blocks and hot-air thermocyclers.
[c] Programmable Heat Blocks. Most thermocyclers resemble “waffle irons.” They are heat blocks with holes in them where plastic reaction tubes are heated and cooled under electronic control. Several such devices have been described by Johnson (1998). The problem with this type of design is that one spends most of ones' time waiting for a block of metal to heat up or cool down. Like the waffle chef—who spends most of his time heating up the waffle iron beforehand, or cooling it off afterwards—very little time is spent actually cooking the waffles. For example, in the MJ Research PTC-150 thermocycler (Watertown, Mass.), 14 seconds/cycle is lost in transition between D, A, and E temperatures (~94° C., 55°, and 72°).
Many commonly employed PCR protocols spend one minute at 94° C. (denaturation), one minute at ~55° C. (annealing), and one minute at 72° C. (elongation). For example, in the original PCR method used by Cetus workers (Saiki et al., 1989b), a 536 b.p. &bgr;-globin DNA fragment was amplified using 30 cycles of (1 min at 94° C., 1 min at 55° C., 1 min at 72° C.). The active duty time for this thermocycling protocol is only ~3.5 minutes=210 seconds. This is the time needed to enzymatically copy a 536 b.p. template 30 times at an elongation rate of ~80 nucleotides/sec (Innis et al., 1988; Gelfand and White, 1990).
Commercially available heat block thermocyclers (Perkin-Elmer, Ericomp, MJ Research, Eppendorf, Techne, BioRad, Snark Technologies) require 20 to 25 seconds to cool from 94° C. to 55° C. and another 14 to 20 seconds to heat from 55° C. to 94° C. (Johnson, 1998). Therefore, the “dead time” for each PCR cycle is another 40±5 seconds per cycle. As shown in
FIG. 4
, commonly employed thermocycling protocols require (220 seconds/cycle×30 cycles)=6600 seconds=110 minutes (Saiki et al., 1989b). Only ~3.5 minutes of this ~2 hours is productively focused on the PCR process.
[d] Hot-Air Thermocyclers. In order to overcome the long transitional dead times of heat blocks, hot-air thermocyclers have been constructed which allow 30 cycles of PCR amplification to be carried out in as little as ~10 to 30 minutes. Wittwer and his colleagues have carried out considerable engineering groundwork to optimize rapid DNA amplification in hot-air PCR thermocyclers (Wittwer et al., 1989; Wittwer and Garling, 1991; Wittwer et al., 1994). The rate-limiting step in the three-step PCR reaction sequence (denaturation/annealing/elongation) is the rate of DNA Polymerase elongation. At an elongation rate of 80 nucleotides/sec by Taq Polymerase (Innis et al., 1988; Gelfand and White, 1990), less than one second per cycle is actually needed to amplify DNA fragments shorter than 100 b.p. using ~20 mer primers. For example, only about five seconds per cycle were needed to copy a 536 b.p.
Nelson R. Michael
Quintanar Andre
McKee Voorhees & Sease, P.L.C.
Sisson B. L.
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