Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-05-15
2002-05-21
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
06391544
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to nucleic acid amplification reactions and in particular relates to amplification reactions that employ a pair of primer sequences to generate copies of a target sequence.
BACKGROUND OF THE INVENTION
Nucleic acid amplification reactions are well known and are employed to increase the concentration of a target nucleic acid in a test sample. The “target nucleic acid” typically is present in a sample in low concentrations and therefore cannot easily be detected without amplifying it to increase the concentration of the target sequence in the sample. The polymerase chain reaction (PCR) is one nucleic acid amplification reaction commonly employed for purposes of amplifying a target nucleic acid sequence.
According to the principles of PCR, “primer sequences” are used to prime synthesis of copies of the target sequence. Specifically, under appropriate conditions, primer sequences hybridize to opposite strands of a double stranded nucleic acid sequence such that the primers flank the target sequence. Once hybridized, the primers are extended using enzymes such as, for example, DNA polymerase which extend the primer sequences to thereby generate copies of the target sequence. Additional copies of the target sequence are generated by cycling the above steps of (i) hybridizing and extending the primer sequences and (ii) dissociating the extended primer sequences (or copies of the target sequence) so that additional primers can hybridize to the original target, as well as copies of the target sequence. Hence, multiple copies of the target sequence are generated.
Once amplified, copies of the target sequence can be detected to determine if the target sequence originally was present in the test sample. Of course, if the target sequence was not present, amplification should not occur and the target sequence should not be detected. In any event, amplified target sequences are typically detected using labels. Labels are moieties that have a detectable property and can be incorporated into the copies of the target sequence. Labels typically are incorporated into the amplified target sequences by attaching the labels to primer sequences that are then incorporated into the amplification product as specified above. Alternatively, for example, extension products can be labeled by incorporating labeled nucleotides into such products during primer extension. The presence of the target sequence in the test sample can then be determined by detecting the labeled amplification product.
Amplified target sequences also can be detected using labeled probes that hybridize to a strand or both strands of an amplified target sequence. However, it is sometimes desirable to employ a probe that hybridizes to only one strand of a double stranded amplification product. The effect of such a detection scheme, at least as it applies to a double stranded target sequence, is that a single strand of amplified target sequence is detected to determine the presence of the target sequence in the test sample. However, detecting a single strand of an amplification product can be inefficient insofar as the signal plateaus and sometimes drops (or hooks) as the number of target sequences originally present in the test sample increases. Alleviating the “hooking” or “plateauing” phenomenon and providing a linear signal over a broader range of target sequence concentrations would be beneficial, especially for amplification based assays designed to quantify the amount of a target sequence in a test sample.
It would be expected that substantially increasing the concentration of one primer over the other would alleviate this problem by generating more of the sequence that is detected. Indeed, U.S. Pat. No. 5,066,584 describes a method for preferentially generating one strand of a double stranded target sequence by vastly increasing the concentration of one primer. However, this requires excess reagents and therefore excess costs associated with preferentially producing one of two single strands. Additionally, substantially increasing primer concentrations may increase the chances of non-specific priming and therefore amplification of non-target sequences. Moreover, many times, competing non-specific reactions will interfere with the efficient amplification of the sequence of interest. Therefore, it may be expected that substantially increasing the concentration of one primer over the other primer may present problems in amplification assays designed to be of high sensitivity (i.e. designed to detect low numbers of a sequence of interest).
SUMMARY OF THE INVENTION
The present invention provides a method of detecting a target sequence in a test sample. The method comprises the steps of: (a) forming a reaction mixture comprising a test sample, amplification reagents, a first primer, and a second primer such that the concentration of the first primer in the reaction mixture is 15% to 250% percent greater than the concentration of the second primer; (b) amplifying the target sequence to generate copies of the target sequence comprising an amplification product from the first and second primers; (c) hybridizing a probe to the amplification product from the first primer to form a hybrid complex; and (d) detecting the hybrid complex as an indication of the presence of the target sequence in the test sample. Preferably, the hybrid complex is detected using labels that can either be directly detectable or indirectly detectable.
Also provided is an improved method for amplifying and detecting a target nucleic acid sequence in a test sample comprising the steps of: (a) forming an amplification mixture comprising a test sample, a first and a second primer sequence, and amplification reagents, (b) amplifying the target sequence to generate copies of the target sequence comprising an amplification product from the first and second primers; and (c) detecting the copies of the target sequence as an indication of the presence of the nucleic sequence in the test sample; wherein the improvement comprises providing the first primer sequence in 15% to 250% excess over the second primer and wherein a probe is hybridized to the amplification product from the first primer to form a hybrid complex and the hybrid complex is detected as an indication of the presence of the nucleic acid sequence in the test sample.
Kits for performing the methods of the invention are also provided.
DETAILED DESCRIPTION OF THE INVENTION
Under appropriate conditions, a primer pair will generate copies of a target sequence in the form of a double stranded amplification product. Unfortunately, however, when a single strand of a double stranded amplification product is detected with a probe, the resulting signal can plateau, or even hook, as the concentration of the original target sequence increases. To a certain extent, this phenomenon is counterintuitive since increasing the concentration of the original target sequence should yield a greater concentration of end product, and therefore, a greater signal should be detected. However, as mentioned above, the resulting signal can plateau. While not wishing to be bound by theory, the hooking effect may be attributable to the presence of higher concentrations of longer product strands driving product strand re-annealing to the exclusion of probe/target strand annealing. Applicants have surprisingly and unexpectedly discovered that the plateauing or hooking phenomenon could be alleviated by increasing the concentration of one primer so that it is slightly higher than the concentration of the other primer.
The method provided herein can be applied to any amplification reaction where a pair of primer sequences is employed to generate double stranded amplification products and only one strand of the double stranded products is detected. The method comprises a step where an amplification mixture is formed. The amplification mixture generally will comprise (i) a test sample, (ii) amplification reagents and (iii) a first and second primer (collectively referred to as a “primer pair
Carrino John J.
Salituro John A.
Abbott Laboratories
Schodin David J.
Whisenant Ethan C.
Yasger Paul D.
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