Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2000-05-23
2002-07-23
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S006120, C435S091200, C435S286200, C435S286400, C435S287300, C435S288400, C435S303100, C435S305300, C435S809000, C422S063000, C422S072000, C422S105000, C422S105000, C422S105000, C436S047000, C204S453000, C204S604000
Reexamination Certificate
active
06423536
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to a method and apparatus for performing small scale reactions. In particular, the instant disclosure pertains to small scale cycling reactions, isothermal reactions, and devices for assembly of sub-microliter reaction mixtures.
BACKGROUND OF THE INVENTION
The Human Genome Program is a scientific endeavor that is a national priority of the United States. The original goal of the federally funded U.S. effort had been to complete the sequence at ten-fold coverage by the year 2005. A draft five-fold deep version of the human genome will now be produced by the year 2001. To accomplish this goal, the effort has accelerated to improve sequencing throughput rates and reduce DNA sequencing costs.
In the late 1970s, Sanger et al. developed an enzymatic chain termination method for DNA sequence analysis that produces a nested set of DNA fragments with a common starting point and random terminations at every nucleotide throughout the sequence. Lloyd Smith, Lee Hood, and others modified the Sanger method to use four fluorescent labels in sequencing reactions enabling single lane separations. This resulted in the creation of the first automated DNA sequencers that used polyacrylamide slab gels. More recently, fluorescent energy-transfer dyes have been used to make dye sets that enhance signals by 2- to 10-fold and simplify the optical configuration.
Automated fluorescent capillary array electrophoresis (CAE) DNA sequencers appear to be the consensus technology to replace slab gels. Capillary gel electrophoresis speeds up the separation of sequencing products and has the potential to dramatically decrease sample volume requirements. The 96-channel CAE instrument, MegaBACE™, which is commercially available from Molecular Dynamics (Sunnyvale, Calif.), uses a laser-induced fluorescence (LIF) confocal fluorescence scanner to detect up to an average of about 625 bases per capillary (Phred 20 window) in 90 minute runs with cycle times of two hours. Confocal spatial filtering results in a higher signal-to-noise ratio because superfluous reflections and fluorescence from surrounding materials are eliminated before signal detection at the photomultiplier tube (PMT). Accordingly, sensitivity at the level of subattomoles per sequencing band is attainable. Confocal imaging is also particularly important in capillary electrophoresis in microchip analysis systems where the background fluorescence of a glass or plastic microchip may be much higher than that of fused silica capillaries. Capillary array electrophoresis systems will solve many of the initial throughput needs of the genomic community for DNA analysis. However, low volume sample preparation still presents a significant opportunity to increase throughput and reduce cost.
While fluorescent DNA sequencers are improving the throughput of DNA sequence acquisition, they have also moved the throughput bottleneck from sequence acquisition back towards sample preparation. In response, rapid methods for preparing sequencing templates and for transposon-facilitated DNA sequencing have been developed as have magnetic bead capture methods that eliminate centrifugation. Thermophilic Archae DNA polymerases have been screened and genetically engineered to improve fidelity, ensure stability at high temperatures, extend lengths, and alter affinities for dideoxynucleotides and fluorescent analogs. These improvements have resulted in lower reagent costs, simpler sample preparation, higher data accuracy, and increased readlengths.
The sequencing community has also developed higher throughput methods for preparing DNA templates, polymerase chain reaction (PCR) reactions, and DNA sequencing reactions. Sample preparation has been increasingly multiplexed and automated using 96- and 384-well microtiter plates, multi-channel pipettors, and laboratory robotic workstations. In general, these workstations mimic the manipulations that a technician would perform and have minimum working volumes of about a microliter, although stand-alone multi-channel pipettors are being used to manipulate smaller volumes.
A typical full-scale sample preparation method for DNA shotgun sequencing on capillary systems begins by lysing phage plaques or bacterial colonies to isolate subcloned DNA. Because capillary electrophoresis is more sensitive to impurities in sequencing reactions than slab gels, the subcloned DNA insert is frequently PCR-amplified to exponentially increase its concentration in the sample. Next, exonuclease I (ExoI) and arctic shrimp alkaline phosphatase (SAP) are added to perform an enzymatic cleanup reaction to remove primer and excess dNTPs that interfere with cycle sequencing. ExoI is used to degrade the single-stranded primers to dNMPs without digesting double-stranded products. SAP converts dNTPs to dNMPs and reduces the DNTP concentration from 200 &mgr;M, as used for the PCR reaction, to less than 0.1 &mgr;M for use with fluorescent sequencing. The reaction is performed at 37° C. and then heated to 65° C. to irreversibly denature the ExoI and SAP.
Because the PCR amplification produces excess template DNA for cycle sequencing, the ExoI/SAP treated PCR sample can be diluted five-fold before cycle sequencing. This reduces the concentration of contaminants into a range that causes less interference with CAE analysis. Cycle sequencing reagents are added, typically with fluorescently labeled dye primers or terminators and the reaction is thermal cycled to drive linear amplification of labeled fragments. Finally, after cycling, the samples are ethanol precipitated, resuspended in formamide, another denaturant, or water, and the sample is electrokinetically injected into the CAE system.
This workflow has resulted in a dramatic improvement in the performance of the MegaBACE system and similar workflows currently appear to be the methods of choice for other CAE systems as well. Using actual samples from single plaques and colonies of human genomic random subclones or Expressed Sequence Tags (ESTs), this workflow with linear polyacrylamide as a separation matrix has improved the success rate of samples over 200 base pairs from about 60% to 85-90%, and has improved the average readlength from about 400 to greater than 600 bases. Furthermore, this method has proven to be quite robust.
While the above sample preparation methods have greatly increased throughput, the cost of reagents remains a major component of the cost of sequencing. CAE requires only subattomoles of sample, but presently samples are prepared in the picomole range. Reducing the reaction volume will therefore reduce the cost of DNA sequencing and still provide enough material for analysis. However, substantial reductions in reaction volume can only be achieved if satisfactory methods can be developed for manipulating and reacting samples and reagents. Ideally, such a method would be automated and configured in order that multiple samples could be produced at one time. Moreover, it would be desirable to integrate such a method as a module capable of interfacing with additional components, such as CAE and a detector for separation and analysis.
Several devices have been designed to aid in the automation of sample preparation. For example, U.S. Pat. No. 5,720,923 describes a system in which small scale cycling reactions take place in tubes with diameters as small as 1 mm. The tubes are subsequently exposed to thermal cycles produced by thermal blocks to effect the desired reaction. Multiple samples may be processed in a single tube by drawing in small amounts of sample, each of which are separated in the tube by a liquid which will not combine with the sample. Fluid moves through the tubes by means of a pump. These features are incorporated into a system which automatically cleans the tubes, moves sample trays having sample containing wells, and brings the tubes into contact with the wells in the sample trays.
U.S. Pat. No. 5,785,926 discloses a system for transporting small volumes of sample. In this system, at least one capillary tube is used to transport s
Hadd Andrew G.
Hellman Bo E. R.
Jovanovich Stevan B.
Roach David J.
Beisner William H.
Molecular Dynamics, Inc.
Schneck David M.
Schneck Thomas
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