Chip-based isothermal amplification devices and methods

Details Chip-based isothermal amplification devices and methods Chip-based isothermal amplification devices and methods

1997-11-19

2002-04-30

Marschel, Ardin H. (Department: 1631)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S091100, C436S501000

Reexamination Certificate

active

06379929

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of molecular biology, and relates to methods for amplifying nucleic acid target sequences in microfabricated devices. It particularly relates to isothermal methods for amplifying nucleic acid targets in microfabricated devices. The present invention also relates to methods of detecting and analyzing nucleic acids in microfabricated devices.
2. Description of Related Art
In vitro nucleic acid amplification techniques have provided powerful tools for detection and analysis of small amounts of nucleic acids. The extreme sensitivity of such methods has lead to their development in the fields of diagnosis of infectious and genetic diseases, isolation of genes for analysis, and detection of specific nucleic acids as in forensic medicine.
Nucleic acid amplification techniques may be grouped according to the temperature requirements of the procedure. Certain nucleic acid amplification methods, such as the polymerase chain reaction (PCR™—Saiki et al., 1985), ligase chain reaction (LCR—Wu et al., 1989; Barringer et al., 1990; Barony, 1991), transcription-based amplification (Kwoh et al., 1989) and restriction amplification (U.S. Pat. No. 5,102,784), require temperature cycling of the reaction between high denaturing temperatures and somewhat lower polymerization temperatures. In contrast, methods such as self-sustained sequence replication (3SR; Guatelli et al., 1990), the Q&bgr; replicase system (Lizardi et al., 1988), and Strand Displacement Amplification (SDA—Walker et al., 1992a, 1992b; U.S. Pat. No. 5,455,166) are isothermal reactions that are conducted at a constant temperature, which is typically much lower than the reaction temperatures of temperature cycling amplification methods.
The SDA reaction initially developed was conducted at a constant temperature between about 37° C. and 42° C. (U.S. Pat. No. 5,455,166). This was because the exo
−
klenow DNA polymerase and the restriction endonuclease (e.g., HindII) are mesophilic enzymes that are thermolabile (temperature sensitive) at temperatures above this range. The enzymes that drive the amplification are therefore inactivated as the reaction temperature is increased.
Methods for isothermal Strand Displacement Amplification, which may be performed in a higher temperature range than conventional SDA (about 50° C. to 70° C., “thermophilic SDA”), were later developed. Thermophilic SDA is described in European Patent Application No. 0 684 315 and employs thermophilic restriction endonucleases that nick the hemimodified restriction endonuclease recognition/cleavage site at high temperature and thermophilic polymerases that extend from the nick and displace the downstream strand in the same temperature range.
Photolithographic micromachining of silicon has been used to construct high-throughput integrated fluidic systems for a variety of chemical analyses. This technology is of particular interest for the development of devices for analysis of nucleic acids, as in their conventional formats such analyses are typically labor- and material-intensive. Ideally, all of the processing steps of the amplification reaction would be conducted on the microfabricated device to produce a completely integrated nucleic acid analysis system for liquid transfer, mixing, reaction and detection that requires minimal operator intervention.
Silicon and glass devices are economically attractive because the associated micromachining methods are, essentially, photographic reproduction techniques. Silicon structures are processed using batch fabrication and lithographic techniques. These processes resemble those of printing where many features may be printed at the same time. These processes permit the simultaneous fabrication of thousands of parts in parallel, thus reducing system costs enormously. Today, silicon fabrication techniques are available to simultaneously fabricate micrometer and submicrometer structures on large-area wafers (100 cm
2
), yielding millions of devices per wafer and may be used to process either silicon or glass substrates.
These characteristics have led to the proposal of silicon and glass as a candidate technology for the construction of high-throughput DNA analysis devices (Woolley and Mathies, 1994; Northrup et al., 1993; Effenhauser et al., 1994). As mechanical materials, both silicon and glass have well-known fabrication characteristics (Petersen, 1982). Microfabricated devices for biochemical and fluidic manipulation are undergoing development in many laboratories around the world (Ramsey et al., 1995; McIntyre 1996). Over the past 10 years, a number of microfluidic devices have been developed that allow the construction of miniaturized “chemical reactors.”Individual components of the system such as pumps (Esashi et al., 1989; Zengerle et al., 1992; Matsumoto and Colgate, 1990; Folta et al., 1992); valves (Esashi et al., 1989, Ohnstein et al., 1990; Smits, 1990); fluid channels (Pfahler et al., 1990); chromatographic and liquid electrophoresis separation systems (Terry et al., 1979; Harrison et al., 1992b-g; Manz et al., 1991; Manz et al., 1992) are available. Although an objective of several research groups, complete silicon-fabricated nucleic acids analysis systems are still at the earliest stages of development.
Other components that have been microfabricated which are applicable to nucleic acid analysis include elements for gel electrophoresis (Zeineh and Zeineh, 1990; Heller and Tullis, 1992; Effenhauser et al., 1994; Woolley and Mathies, 1994, 1995; Webster et al., 1996); capillary electrophoresis (Manz et al. 1992, 1995; Effenhauser et al., 1993; Fan and Harrison, 1994; Jacobsen et al., 1994a; 1994b; Jacobson and Ramsey, 1995; Ocvirk et al., 1995; von Heeren et al., 1996); synthetic oligonucleotide arrays (Fodor et al., 1993; Schena et al., 1995; Hacia et al., 1996); continuous flow pumps (Lintel, 1988; Esashi et al., 1989; Matsumoto and Colgate, 1990; Nakagawa et al., 1990; Pfahler et al., 1990; Smits, 1990; Wilding et al, 1994; Olsson et al., 1995); discrete drop pumps (Burns et al., 1996); enzymatic reaction chambers (Northrup et al., 1994; Wilding et al., 1994b; Cheng et al., 1996); optical/radiation detectors (Belau et al., 1983; Wouters and van Sprakelaar, 1993; Webster et al., 1996); and multicomponent systems (Harrison et al., 1992, 1995; Northrup et al. 1994; Jacobsob and Ramsey 1996).
To date, a number of devices have been micromachined, including pumps and valves (Gravensen et al., 1993; Manz et al., 1994; Colgate and Matsumoto, 1990, Sammorco et al, 1996); reaction chambers (Woolley and Mathies, 1994; Wilding et al., 1994); and separation and detection systems (Weber and May, 1989, Northrup et al., 1993, Harrison et al., 1993; Manz et al., 1992; Jacobson et al., 1994; Schoonevald et al., 1991; Van den Berg and Bergveld, 1995; Woolley et al., 1995). Some of these have been recently integrated together to build pharmaceutical drug closing systems (Lammerink et al., 1993; Miyake et al., 1993) and other microchemical systems (Nakagawa et al., 1990; Washizu, 1992; Van den Berg and Bergveld, 1995). One device is an integrated glass system combining DNA restriction enzyme digestion and capillary electrophoresis (Jacobson and Ramsey 1996). An alternative format using high-density arrays of synthesized oligodeoxynucleotides has been demonstrated as a DNA sequence detector (Fodor et al., 1993; Hacia et al., 1996).
Nucleic acid targets have been successfully amplified by the PCR™ on such microfabricated devices, often referred to as “chips” (U.S. Pat. No. 5,498,392; Woolley et al., 1996; Shoffner et al., 1995; Cheng et al., 1996; Wilding et al., 1994; U.S. Pat. No. 5,589,136; U.S. Pat. No. 5, 639,423; U.S. Pat. No. 5,587,128, U.S. Pat. No. 5,451,500) and LCR (Cheng et al., 1996; U.S. Pat. No. 5,589,136). Evaporation due to repeated exposure to high temperatures during thermocycling is a problem. Evaporation during PCR™ has been controlled by immersing the channel in oil such that the open ends are covered

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