Throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.
This invention relates to methods of detecting and quantitating target nucleic acid molecules in a sample via nucleic acid amplification. In the instant methods, a single amplicon is produced containing both catalytic nucleic acid and target sequences. The catalytic nucleic acid is synthesized from its anti-sense, zymogenic precursor only if the target is present.
Methods of in vitro nucleic acid amplification have wide-spread applications in genetics, disease diagnosis and forensics. In the last decade many techniques for amplification of known nucleic acid sequences (xe2x80x9ctargetsxe2x80x9d) have been described. These include the polymerase chain reaction (xe2x80x9cPCRxe2x80x9d) (1-7, 41), the strand displacement amplification assay (xe2x80x9cSDAxe2x80x9d) (8) and transcription-mediated amplification (xe2x80x9cTMAxe2x80x9d) (9, 10) (also known as self-sustained sequence replication (xe2x80x9cSSRxe2x80x9d)). The amplification products (xe2x80x9campliconsxe2x80x9d) produced by PCR and SDA are DNA, whereas RNA amplicons are produced by TMA. The DNA or RNA amplicons generated by these methods can be used as markers of nucleic acid sequences associated with specific disorders.
Several methods allow simultaneous amplification and detection of nucleic acids in a closed system, i.e., in a single homogeneous reaction system. These methods include Sunrise(trademark) primers (11), Molecular Beacons (12) and the Taqman(trademark) system (13). Using homogeneous sealed tube formats has several advantages over separately analyzing amplicons following amplification reactions. Closed system methods are faster and simpler because they require fewer manipulations. A closed system eliminates the potential for false positives associated with contamination by amplicons from other reactions. Homogeneous reactions can be monitored in real time, with the signal at time zero allowing the measurement of the background signal in the system. Additional control reactions for estimating the background signal are therefore not required. A change in the signal intensity indicates amplification of a specific nucleic acid sequence present in the sample.
Instead of amplifying the target nucleic acid, alternate strategies involve amplifying the reporter signal. The Branched DNA assay (14) amplifies the signal by employing a secondary reporter molecule (e.g. alkaline phosphatase), whereas fluorescence correlation spectroscopy (FCS) employs electronic amplification of the signal (15).
As with other amplification technologies, catalytic nucleic acids have been studied intensively in recent years. The potential for suppression of gene function using catalytic nucleic acids as therapeutic agents is widely discussed in the literature (16-22). Catalytic RNA molecules (xe2x80x9cribozymesxe2x80x9d) have been shown to catalyze the formation and cleavage of phosphodiester bonds (16, 23). In vitro evolution techniques have been used to discover additional nucleic acids which are capable of catalyzing a far broader range of reactions including cleavage (21, 22, 24) and ligation of nucleic acids (25), porphyrin metallation (26), and formation of carbon-carbon (27), ester (28) and amide bonds (29).
Ribozymes have been shown to be capable of cleaving both RNA (16) and DNA (21) molecules. Similarly, catalytic DNA molecules (xe2x80x9cDNAzymesxe2x80x9d) have also been shown to be capable of cleaving both RNA (17, 24) and DNA (22, 30) molecules. Catalytic nucleic acid can cleave a target nucleic acid substrate provided the substrate meets stringent sequence requirements. The target substrate must be complementary to the hybridizing regions of the catalytic nucleic acid and contain a specific sequence at the site of cleavage. Examples of sequence requirements at the cleavage site include the requirement for a purine:pyrimidine sequence for a class of DNAzymes (xe2x80x9c10-23 modelxe2x80x9d or xe2x80x9c10-23 DNAzymexe2x80x9d) (24), and the requirement for the sequence U:X where X can equal A, C or U but not G, for hammerhead ribozymes (16).
In addition to having therapeutic potential, catalytic nucleic acid molecules can also be used as molecular tools in genetic diagnostic assays. For example, ribozymes have been used to facilitate signal amplification in a two-stage method (31-33). In the first stage, a test sample is contacted with inactive oligonucleotides. This contacting results in the production of xe2x80x9ctriggeringxe2x80x9d RNA oligonucleotides when the sample contains the target sequence. In the second stage the triggering RNA oligonucleotides induce an amplification cascade. This cascade results in the production of large quantities of catalytically active reporter ribozymes which, when detected, indicate the presence of the target sequence in the test sample. The target sequence itself is not amplified during the process. Rather, only the reporter signal is amplified.
In short, target nucleic acid amplification and reaction conditions permitting same are known. Catalytic nucleic acid molecules, and reaction conditions permitting their activity are also known.
However, no method has ever existed which permits the simultaneous processes of nucleic acid amplification and catalytic nucleic activity in a single reaction milieu. Moreover, no target amplification method has ever been performed wherein the amplification product is a single nucleic acid molecule containing sequences for the target and the catalytic nucleic acid molecule. Finally, no target amplification method has ever employed an anti-sense, zymogenic sequence of a catalytic nucleic acid molecule which, only in the presence of target sequence, is amplified in its xe2x80x9csensexe2x80x9d, catalytic form.
This invention provides a method of detecting the presence of a target nucleic acid sequence in a sample which comprises
(a) contacting the sample, under conditions permitting primer-initiated nucleic acid amplification and catalytic nucleic acid activity, with
(i) a DNA primer suitable for initiating amplification of the target, and
(ii) a DNA zymogene which encodes, but which itself is the anti-sense sequence of, a catalytic nucleic acid molecule, wherein the primer and zymogene are situated with respect to each other so that, when the target is present, a single amplified nucleic acid molecule is produced which comprises the sequences of both the target and catalytic nucleic acid molecule; and
(b) determining the presence of catalytic nucleic acid activity, thereby determining the presence of the target nucleic acid sequence in the sample.
This invention also provides a method of simultaneously detecting the presence of a plurality of target nucleic acid sequences in a sample which comprises
(a) contacting the sample, under conditions permitting primer-initiated nucleic acid amplification and catalytic nucleic acid activity, with
(i) a plurality of primers wherein for each target being detected, there exists at least one primer suitable for initiating amplification of that target, and
(ii) a plurality of zymogenes wherein for each target being detected, there exists at least one zymogene which encodes, but which itself is the anti-sense sequence of, a catalytic nucleic acid molecule having distinctly measurable activity, the primer and zymogene being situated with respect to each other so that, when the corresponding target is present, a single amplified nucleic acid molecule is produced which comprises the sequences of both the target and corresponding catalytic nucleic acid molecule; and
(b) simultaneously determining the presence of each of the catalytic nucleic acid activities, thereby determining the presence of each of the corresponding target nucleic acid sequences in the sample.
This invention further provides a DNA molecule comprising a primer and a zymogene, wherein the primer is situated 3xe2x80x2 of the zymogene.
This invention still further provides a kit for use in determining the presence of a target nucleic acid sequence in a sample, which comprises
(a) a primer suitable for initiating amplification of the target;
(b) a zymogene which encodes, but which itself is the anti-sense sequence of, a catalytic nucleic acid sequence, wherein the primer and zymogene are situated with respect to each other so that, when the target is present, a single amplified nucleic acid molecule is produced which comprises the sequences of both the target and catalytic nucleic acid molecule; and
(c) reagents permitting primer-initiated nucleic acid amplification and catalytic nucleic acid activity.
Finally, this invention provides a kit for use in determining the presence of a plurality of target nucleic acid sequences in a sample, which comprises
(a) a plurality of primers, wherein for each target being detected, there exists at least one primer suitable for initiating amplification of that target;
(b) a plurality of zymogenes wherein for each target being detected, there exists at least one zymogene which encodes, but which itself is the anti-sense sequence of, a catalytic nucleic acid sequence having distinctly measurable activity, wherein the primer and zymogene are situated with respect to each other so that, when the corresponding target is present, a single amplified nucleic acid molecule is produced which comprises the sequences of both the target and corresponding catalytic nucleic acid molecule; and
(c) reagents permitting primer-initiated nucleic acid amplification and catalytic nucleic acid activity.