Pressure-controlled nucleic acid hybridization

Details Pressure-controlled nucleic acid hybridization Pressure-controlled nucleic acid hybridization

2001-07-09

2004-06-22

Siew, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C435S006120, C435S007100, C435S091100, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330

Reexamination Certificate

active

06753169

ABSTRACT:

FIELD OF THE INVENTION
The invention is in the general field of nucleic acid hybridization.
BACKGROUND OF THE INVENTION
Nucleic acid hybridization is an important technique for detecting the presence of particular sequence information. Recent advances have allowed for the production of high density oligonucleotide arrays which may have great utility for research and clinical diagnostics. Typically, a sequence is amplified and labeled with fluorescent tags. The sample is incubated with the probe array and the sample is washed with a series of increasingly stringent buffers. Stringency may be provided by means of reduced salt concentration or increased temperature. Reduced salt concentrations lead to electrostatic repulsion between phosphate groups, thereby lowering the melting temperature (T
m
).
The number of different sequences on each array is ultimately limited by the detection capabilities of the instrument analyzing it and factors such as contaminating fluorescent compounds, stray light, and hybridization of mismatched oligonucleotides. As the stringency of hybridization is increased, the noise level from mismatches is reduced (increasing specificity), but the overall signal is also reduced due to loss of correctly hybridized molecules (decreasing sensitivity).
Two related and important amplification techniques which depend on the specificity and sensitivity of oligonucleotide hybridization are polymerase chain reaction (PCR) and ligase chain reaction (LCR), which are commonly used in medical diagnostics and research.
SUMMARY OF THE INVENTION
The invention relates to controlling the specificity, sensitivity, or selectivity of nucleic acid hybridization procedures. Control of hybridization according to the present invention is achieved through application of high hydrostatic pressure. Without intending to be limited to any mechanism, it is believed that increased pressure favors nucleic acid hybridization (i.e., reversible or irreversible hybridization). Regardless of the mechanism involved, high pressure increases the sensitivity, specificity, or selectivity of nucleic acid hybridizations.
Accordingly, the invention features a method of hybridizing a first nucleic acid to a second nucleic acid at least partially complementary to the first nucleic acid by (1) providing a sample vessel and pressure controller for the vessel; and (2) contacting the first and second nucleic acids within the vessel at a pressure above ambient pressure (e.g., above 10,000 psi) which is effective to enhance hybridization of the first and second nucleic acids. This method opitionally includes cycling pressure in the vessel between a first higher pressure at which the first and second nucleic acid are hybridized and a second lower pressure at which the first and second nucleic acid are denatured. In case of pressure cycling, can further include providing a temperature control for the sample vessel, and cycling the temperature between a lower temperature and a higher temperature, such that the first and second nucleic acids hybridize at the first pressure and lower temperature, and such that the first and second nucleic acids denature at the second pressure and higher temperature. Alternatively, the vessel can be maintained at a constant temperature as the pressure is cycled. Such methods can be used to amplify a portion of the second nucleic acid. An optional step in any of the above methods includes washing away unhybridized nucleic acids after increasing the pressure but before decreasing the pressure.
In another embodiment, the invention features a method of detecting in a sample the presence of a nucleic acid that hybridizes to a reference nucleic acid at a first higher pressure but not at a second lower pressure by (1) providing a sample vessel and pressure controller for the vessel; and in any order (2) contacting the reference sequence with the sample in the vessel at the first pressure; (3) contacting the reference sequence with the sample in the pressure vessel at the second pressure; and (4) detecting the presence of a nucleic acid that hybridizes to the reference nucleic acid at the first pressure but not at the second pressure. In one aspect, the reference sequence is first contacted with the sample and hybridization is detected, and then the pressure is lowered and the absence of hybridization is detected.
The invention also features a method of discriminating between a first nucleic acid and a second nucleic acid that is different from the first nucleic acid by (1) providing a sample vessel and pressure controller for the vessel; (2) maintaining the vessel at a constant pressure; (3) providing the first and second nucleic acid and a reference nucleic acid in the vessel under conditions that do not allow either the first or the second nucleic acid to hybridize to the reference nucleic acid; (4) perturbing at least one condition (e.g., temperature or an electric field) to establish conditions that permit the first nucleic acid to form a complex with the reference nucleic acid at equilibrium and to permit the second nucleic acid to form a complex with the reference nucleic acid at equilibrium; and (5) comparing the time necessary to achieve equilibrium hybridization between the first nucleic acid and the reference nucleic acid with the time necessary to achieve equilibrium hybridization between the second nucleic acid and the reference nucleic acid, wherein the difference indicates the relative difference in sequence between the first and the second nucleic acids.
In addition, the invention features a method of discriminating between a first nucleic acid and a second nucleic acid that is different from the first nucleic acid by (1) providing a sample vessel and pressure controller for the vessel; (3) providing the first and second nucleic acid and a reference nucleic acid in the vessel under a first pressure that does not allow either the first or the second nucleic acid to hybridize to the reference nucleic acid; (4) perturbing the pressure to establish conditions that permit the first nucleic acid to form a complex with the reference nucleic acid at equilibrium and to permit the second nucleic acid to form a complex with the reference nucleic acid at equilibrium; and (5) comparing the time necessary to achieve equilibrium hybridization between the first nucleic acid and the reference nucleic acid with the time necessary to achieve equilibrium hybridization between the second nucleic acid and the reference nucleic acid, wherein the difference indicates the relative difference in sequence between the first and the second nucleic acids.
Methods of placing nucleic acids and optionally enzymes under pressure are described in WO 96/27432.
To facilitate understanding of the invention, a number of terms are defined below.
As used herein, the term “solution” refers to a liquid, and more particularly, the incorporation of substances (e.g. dissolved compounds) in a liquid. The term “aqueous solution” refers to a solution either containing water or that is like water. For example, the present invention contemplates the use of assay buffers that are aqueous solutions.
As used herein, the term “vector” is used in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The term “vehicle” is sometimes used interchangeably with “vector.”
The term “expression vector” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
As used herein, the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence

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