Non-competitive co-amplification methods

Details Non-competitive co-amplification methods Non-competitive co-amplification methods

2000-10-20

2002-10-08

Whisenant, Ethan C. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S023100, C536S024300

Reexamination Certificate

active

06461817

ABSTRACT:

This invention relates to amplification assays to detect nucleic-acid targets.
BACKGROUND OF THE INVENTION
This application relates to assays employing exponential amplification of a target sequence. The target sequence may be RNA or DNA. By “amplification of a target sequence,” we mean to include amplification of the target sequence itself and also amplification of a transcript thereof, as when an RNA target sequence is amplified by first creating a DNA transcript with reverse transcriptase and then amplifying the DNA transcript. By “exponential amplification” we mean an amplification reaction or reactions that generate products (“amplicons”) that include both plus strands and complementary minus strands.
In referencing target sequences, control sequences, amplicons and probes, we mean to include both plus and minus strands. Thus, it will be understood that when referring to cross hybridization of a control sequence with a target sequence or to cross hybridization of two target sequences during amplification, we are referring to hybridization of the plus strand of one to the minus strand of the other. Similarly, when we refer to hybridization of a probe to a target sequence, we mean to include hybridization of the probe to either the plus-strand or the minus strand of the target sequence itself or to an amplicon, that is a plus-strand copy or minus-strand copy of the target sequence.
Several reaction schemes are used in assays employing amplification of a target sequence. The most widely used is the polymerase chain reaction (PCR) process. It will be used herein for presentation of the specifics of the prior art and the specifics of this invention. However, it will be understood that this invention also applies to other reaction schemes, including nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), self-sustained sequence replication (
3
SR) (Guatelli et al. (1990)) and strand displacement amplification (SDA) (Walker et al. (1992)).
The polymerase chain reaction process is well known. It is the most widely used technique for amplifying DNA and RNA (RT-PCR) targets, including amplification as part of assays to detect the presence of DNA and RNA targets for many purposes, including, for example, in vitro diagnostics, genetic analyses, forensics, food and agricultural testing, and parentage testing. PCR is used for detection even at the level of a single cell (in situ PCR).
Quantitative PCR assays are also well known. Quantitative PCR assays for DNA and RNA have been widely used to study disease processes (see, for example, Clementi et al., 1993). One type of quantitative PCR assay involves simultaneously amplifying control molecules and samples containing (or suspected to contain) a target sequence. Receptacles containing known amounts of control molecule are thermally cycled with receptacles, most commonly tubes or wells or slides, containing the unknown amount of target. In addition to the pair of PCR primers for the target, a pair of PCR primers is required for each control molecule. Following amplification, the amounts of amplified products (amplicons) are compared. See generally Clementi el al., 1994 and Kahn et al., 1992. Partly due to variation in amplification efficiency among primers, only relative quantitation between samples is possible.
Another type of quantitative PCR assay is quantitative-competitive PCR (QC PCR). In this method a control molecule which is similar to but ultimately distinguishable from the target sequence competes with the target sequence for the same pair of primers. Following competitive amplification, the two products synthesized (amplicons) are distinguished, for example, by size using gel electrophoresis. See generally Wang et al., 1989 and Becker-Andre, 1991. While permitting more than relative quantitation between samples, QC PCR has inherent disadvantages and limitations. Post-amplification manipulation is required. This complicates the assay, decreases throughput, increases labor, and risks contamination of untested samples by amplicon carryover. Assay design is complicated by the need for a competitor-control that amplifies with an efficiency very close to that of the target unknown. For reasonable quantitation, most QC PCR assays are performed in multiple tubes containing serial dilutions, of the competitor-control, typically five-fold dilutions, but in some assays two-fold dilutions for better accuracy. Differences in amplification efficiency between the target and the competitor-control usually compel analysis during the exponential phase of amplification, because errors become too large during the subsequent linear phase. (Mullis and Faloona, 1987). Precision is limited, varying a minimum of fifty percent between parallel assays.
A more recently developed type of quantitative PCR assay has been called the 5′-nuclease assay and “real-time PCR.” See generally, Gibson et al., 1996; Heid et al., 1996; Gelfand et al., 1993; and Livak et al., 1996. This method utilizes detector probes that are linear DNA sequences labeled with two different fluorescent dyes, for example, a reporter dye such as FAM and a quenching dye such as TAMRA. Commercial kits from the Applied Biosystems Division of The Perkin-Elmer Corporation (Foster City, Calif. (U.S.A.)) are available under the trademark TaqMan™. When not hybridized to target (original unknown or amplicon) the quenching dye partially quenches the reporter dye. During the annealing step of a PCR cycle, the probes hybridize to the target sequence, and during the extension step of the PCR cycle, the probes are cleaved by the 5′→3′ nucleolytic activity of DNA polymerase. Cleavage releases the reporter dye from the quenching dye, resulting in an increase in fluorescence. Fluorescence can be monitored throughout the PCR amplification. An instrument available from the Applied Biosystems Division of The Perkin-Elmer Corporation, the ABI PRISM 7700, monitors fluorescence in 96 tubes simultaneously in real time. An improved probe suitable for real-time PCR has been developed. See Tyagi and Kramer, 1996. This probe, referred to as a “molecular beacon”, possesses a stem-and-loop structure, has a higher signal-to-background ratio than linear probes and also has improved allele discrimination. During the annealing step of a PCR cycle, molecular beacon probes hybridize to the target sequence and fluoresce, but during the extension step of the PCR cycle, the probes leave the target and do not interfere with polymerization.
Due to sample-to-sample variations in PCR efficiency, only data from the early, exponential amplification phase should be used. That limited data permits a determination of the PCR cycle number at which fluorescence becomes detectable above background (the cycle threshold). The cycle threshold decreases in proportion to the logarithm of initial target concentration. A standard curve can be generated from the cycle thresholds of a dilution series of known starting concentrations of target, and the cycle threshold of a sample containing an unknown amount of target sequence can be compared to the standard curve in order to determine the amount of target sequence present in the sample. Real-time PCR has a wider dynamic range than QC PCR. Importantly, it does not suffer from the serious disadvantages resulting from opening tubes after amplification. It utilizes homogeneous detection with a probe that is added prior to amplification. Nevertheless, accuracy is limited due to variations in amplification efficiency. For example, Gibson et al. (1996) performed real-time PCR using two sets of tubes. Each set contained triplicate two-fold dilutions of a control molecule and a fixed amount of unknown. Despite use of averaged triplicate samples of two-fold dilutions to create a standard curve for cycle thresholds and use of averaged triplicate samples of unknown, quantitation of the unknown in the two separate experiments differed by thirty percent.
SUMMARY OF THE INVENTION
An aspect of this invention is nucleic acid hybridization assays that

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