Electronically mediated nucleic acid amplification in NASBA

Details Electronically mediated nucleic acid amplification in NASBA Electronically mediated nucleic acid amplification in NASBA

1999-04-12

2001-12-04

Horlick, Kenneth R. (Department: 1656)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C436S006000, C436S091000, C436S169000, C436S169000, C436S169000, C436S169000, C530S333000, C530S333000, C530S333000, C530S333000, C530S333000, C530S333000

Reexamination Certificate

active

06326173

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to devices, methods, and compositions of matter for performing active, multi-step, and multiplex nucleic acid sequence separation, amplification and diagnostic analyses. Generally, it relates to devices, methods, and compositions of matter for amplification and analysis of nucleic acid sequences in a sample. More specifically, the invention relates to methods, devices, and compositions of matter for amplifying and analyzing nucleic acids using novel strand displacement amplification technologies in combination with bioelectronic microchip technology. The devices and methods of the invention are useful in a variety of applications, including, for example, disease diagnostics (infectious and otherwise), genetic analyses, agricultural and environmental applications, drug discovery, pharmacogenomics, and food and/or water monitoring and analysis.
BACKGROUND OF THE INVENTION
The following description provides a summary of information relevant to the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to that invention.
Definitions
The following descriptions of the inventions contained herein use numerous technical terms specific to the field of the invention. Generally, the meaning of these terms are known to those having skill in the art and are further described as follows:
As used herein, “sample” refers to a substance that is being assayed for the presence of one or more nucleic acids of interest. The nucleic acid or nucleic acids of interest may be present in a mixture of other nucleic acids. A sample, containing the nucleic acids of interest, may be obtained in numerous ways. It is envisioned that the following could represent samples: cell lysates, purified genomic DNA, body fluids such as from a human or animal, clinical samples, food samples, etc.
As used herein, the phrases “target nucleic acid” and “target sequence” are used interchangeably. Both phrases refer to a nucleic acid sequence, the presence or absence of which is desired to be detected. Target nucleic acid can be single-stranded or double-stranded and, if it is double-stranded, it may be denatured to single-stranded form prior to its detection using methods, as described herein, or other well known methods. Additionally, the target nucleic acid may be nucleic acid in any form most notably DNA or RNA.
As used herein, “amplification” refers to the increase in the number of copies of a particular nucleic acid target of interest wherein said copies are also called “amplicons” or “amplification products”.
As used herein, “amplification components” refers to the reaction materials such as enzymes, buffers, and nucleic acids necessary to perform an amplification reaction to form amplicons or amplification products of a target nucleic acid of interest.
As used herein, the phrase “multiplex amplification” refers to the amplification of more than one nucleic acid of interest. For example, it can refer to the amplification of multiple sequences from the same sample or the amplification of one of several sequences in a sample, as described in U.S. Pat. Nos. 5,422,252 and 5,470,723 which are incorporated herein by reference. The phrase also refers to the amplification of one or more sequences present in multiple samples either simultaneously or in step-wise fashion.
As used herein, “oligonucleotide” refers to a molecule comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than three. The length of an oligonucleotide will depend on how it is to be used. The oligonucleotide may be derived synthetically or by cloning. Oligonucleotides may also comprise protein nucleic acids (PNAs).
As used herein, “probe” refers to a known sequence of a nucleic acid that is capable of selectively binding to a target nucleic acid. More specifically, “probe” refers to an oligonucleotide designed to be sufficiently complementary to a sequence of one strand of a nucleic acid that is to be probed such that the probe and nucleic acid strand will hybridize under selected stringency conditions. Specific types of oligonucleotide probes are used in various embodiments of the invention. For example, a “ligation probe” describes one type of probe designed to bind to both a target nucleic acid of interest and to an amplification probe. Additionally, a “ligated probe” or a “ligated probe template” refers to the end product of a ligation reaction between a pair of ligation probes.
As used herein, the terms “primer molecule” and “primer” are used interchangeably. A primer is a nucleic acid molecule with a 3′ terminus that is either “blocked” and cannot be covalently linked to additional nucleic acids or that is not blocked and possesses a chemical group at the 3′ terminus that will allow extension of the nucleic acid chain such as catalyzed by a DNA polymerase or reverse transcriptase.
As used herein, the phrase “amplification primer” refers to an oligonucleotide primer used for amplification of a target nucleic acid sequence.
The phrase “primer extension,” as used herein refers to the DNA polymerase induced extension of a nucleic acid chain from a free three-prime (3′) hydroxy group thereby creating a strand of nucleic acid complementary to an opposing strand.
As used herein, the term “amplicon” refers to the product of an amplification reaction. An amplicon may contain amplified nucleic acids if both primers utilized hybridize to a target sequence. An amplicon may not contain amplified nucleic acids if one of the primers used does not hybridize to a target sequence. Thus, this term is used generically herin and does not imply the presence of amplified nucleic acids.
As used herein, “electronically addressable” refers to a capacity of a microchip to direct materials such as nucleic acids and enzymes and other amplification components from one position to another on the microchip by electronic biasing of the capture sites of the chip. “Electronic biasing” is intended to mean that the electronic charge at a capture site or another position on the microchip may be manipulated between a net positive and a net minus charge so that charged molecules in solution and in contact with the microchip may be directed toward or away from one position on the microchip or from one position to another position.
As used herein, the phrase “capture site” refers to a specific position on an electronically addressable microchip wherein electronic biasing is initiated and where molecules seen as nucleic acid probes and target molecules are attracted or addressed by such biasing.
As used herein, the term “anchored” refers to the immobilization by binding of a molecule to a specified location on a microchip, such as a primer nucleic acid used in an SDA reaction, or a nucleic acid probe used to capture a target nucleic acid.
As used herein, the term “branched primer pair” refers to a pair of oligonucleotides that may be used as primers in an amplification reaction and which are connected together through a chemical moiety such that the oligonucleotides are susceptible to hybridization and use as amplification primers.
As used herein, the term “primer capture probes” refers to oligonucleotides that are used to hybridize to selected target nucleic acids and provide anchoring support for such nucleic acids to a capture site. Moreover, such oligonucleotides may function as amplification primers for amplifying said target nucleic acids.
As used herein, “hybridization” and “binding” are used interchangeably and refer to the non-covalent binding or “base pairing” of complementary nucleic acid sequences to one another. Whether or not a particular probe remains base paired with a polynucleotide sequence depends on the degree of complementarity, the length of the probe, and the stringency of the binding conditions. The higher the stringency, the higher must be the degree of complementarity, and/or the longer the probe for binding or base

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