Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-07-30
2001-06-12
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S025300, C422S068100, C549S022000, C250S282000
Reexamination Certificate
active
06245506
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention is in the field of devices and methods for sequencing biopolymers.
The prevailing DNA sequencing methods are based on Sanger chemistry and on fragment analysis using gel-based electrophoresis. These methods reveal the base pair sequence of individual fragments of DNA. The bases are separated by subjecting the fragments, suspended in a slab gel, to an electrical field. This causes size-dependent migration and spatial separation of the fragments. Once they have been separated on the gel, the bands corresponding to the individual base pairs are read or digitally scanned to determine the fragment sequence.
Although the results obtained from gel electrophoresis are generally of high quality and reliability, the process is labor intensive and relatively slow. Due to its complexity, gel electrophoresis often requires a skilled technician. Additionally, preparing samples prior to sequencing requires that the target be isolated, purified, amplified, and fragmented into relatively smaller pieces (e.g., about 300 to 500 base pairs). Since the average length of a human gene is over 50,000 bases (i.e., 15,000 to over 1,000,000 bases), considerable sample preparation is necessary to systematically fragment, purify, and amplify the fragments.
To ensure that each fragment is sequenced at least once, the target section is often deliberately overlapped, with the consequence that the same bases may be sequenced ten times or more in the end. Once the sequencing has been completed, the resulting data are processed to deduce the sequence of the original target section.
Improved engineering and automation have resulted in sequencing systems that include such technological advances as automated gel-based electrophoresis or ultra-thin capillary tube electrophoresis. These techniques permit higher speed and lower cost sequencing but are still limited by the fundamental constraints of Sanger-based chemistry and fragment analysis, namely the need for highly trained personnel to prepare relatively short read lengths. Nonetheless, automated gel electrophoresis is the technique currently used for almost all high throughput commercial sequencing. Current efforts toward gene discovery, for example those based on “population-based” genetic analysis, can create tremendous demand for cost-effective DNA sequence analysis. For example, HIV protease inhibitor medications recently introduced rely heavily on DNA sequencing of individual patient samples to detect the emergence of resistant strains of HIV and subsequently alter choice of therapeutic intervention. Indeed, cost-effective DNA sequence analysis methods are likely to prove to be a prerequisite for “individual-based” preclinical and clinical patient studies.
SUMMARY OF THE INVENTION
The invention is based on the discovery that the sequence of monomers in a polymeric biomolecule can be determined in a self-contained, high pressure reaction and detection apparatus, without the need for fluid flow into or out from the apparatus. The pressure is used to control the activity of enzymes that digest the polymeric biomolecule to yield the individual monomers in the sequence in which they existed in the polymer. High pressures modulate enzyme kinetics by reversibly inhibiting those enzymatic processes which result in a higher average activation volume, when compared to the ground state, and reversibly accelerating those processes which have lower activation volumes than the ground state. Modulating the pressure allows the experimenter to precisely control the activity of the enzyme. Conditions can be found, for example, where the enzyme removes only one monomer (e.g., a nucleotide or amino acid) from the biomolecule before the pressure is again raised to a prohibitive level. The identity of the single released nucleotide or amino acid can be determined using a detector that is in communication with a probe in the detection zone within the reaction vessel.
In general, the invention features an integrated device for sequencing a polymeric biomolecule, including: a reaction vessel that includes a reaction zone and a detection zone; a solid support in the reaction zone for chemical attachment of the polymeric biomolecule; an enzyme that catalyzes the removal of one monomer unit at a time from one end of the polymeric biomolecule; a probe for sensing a characteristic (e.g., fluorescence, mass, impedance, optical, voltammetric or amperometric properties, etc.) of the released monomers positioned within the detection zone of the reaction vessel; and a pressure-control device (e.g., piezoelectric crystal-driven pressure modulation, thin-film-driven pressure modulation, electronic, pneumatic, hydraulic, magnetostrictive, etc.) that controls the pressure at least in the reaction zone of the reaction vessel.
Solid supports are available in many configurations, including beads, filters, membranes, capillaries, and frits. Both organic and inorganic supports can be used. For example, sephadex, agarose, dextran, latex, silica gel, glass, polyacrylamide, polystyrene, polyethylene, polyvinylidenefluoride, and other polymers; collagen and similar gels; and biological substrates can all be suitable.
The solid supports can be activated to form specific bonds with biomolecules. Reagents used to activate the solid supports toward covalent bonding include the cyanogen halides, sulfonyl chloride, periodates, sulfonate glutaraldehyde, and carboxyl functionalized compounds.
The reaction zone can be either spatially separated from the detection zone or not.
The probe can be an optical window of quartz or sapphire.
The device can also include a modulated electrophoretic, electroosmotic, fluid flow, or flow cytometry device to connect the reaction and detection zones. The modulation device can contain a buffer, the pH of which is pressure-sensitive.
The device can include multiple probes within its detection zone, allowing it to sense a characteristic of a multiplicity of monomers (e.g., for parallel analysis of multiple single molecule sequencing reactions).
The probe can be, for example, an optical fiber for fluorescence detection, a fluorescence microscope (e.g., CCD-based or confocal), an infrared or Raman spectrometer, a fluorescence polarimeter, an enzymatic biosensor, or a mass spectrometer.
The polymeric biomolecule can be a nucleic acid or a polypeptide, for example.
At least some of the monomers of the polymeric biomolecule can be labelled with fluorescent tags, and the identity of the fluorescently labelled monomers can then be determined by fluorescence resonance energy transfer between the monomers and the enzyme.
Another embodiment of the invention is a sample plate that includes a solid surface adapted for use in the sequencing device described above, and a linker molecule covalently bonded to the surface and to a primer molecule complementary to a biomolecule to be sequenced.
Still another embodiment of the invention features a method for sequencing a polymeric biomolecule. The method includes the steps of immobilizing the polymeric biomolecule on a solid support in a reaction vessel; associating an ion-dependent, biomolecule-digesting enzyme with the nucleic acid under conditions in which the activity of the enzyme is blocked by an exogenously controllable characteristic that can be altered without adding a reagent from a separate vessel; then, with the pressure in the vessel at a level that inactivates the enzyme, altering the exogenously controllable characteristic to allow the enzyme's biomolecule digesting activity to be functional and to excise the terminal monomer from the polymeric biomolecule; adjusting the pressure to activate the enzyme to dissociate one monomer from the polymeric biomolecule; determining the identity of the dissociated terminal monomer with a probe for sensing a characteristic of the dissociated monomer within the reaction vessel; adjusting the pressure to inactivate the enzyme; and repeating the pressure adjusting and identity determining steps. The biomolecule immobilizing and enzyme associating steps
Hess Robert A.
Laugharn, Jr. James A.
BBI BioSeq, Inc.
Fish & Richardson P.C.
Horlick Kenneth R.
Siew Jeffrey
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