The subject matter disclosed herein relates to a polymerase chain reaction (PCR) process and, more particularly, to a method for nested detection of a PCR product.
PCR is a technique that allows for replicating and amplifying trace amounts of DNA fragments into quantities that are sufficient for analysis. As such, PCR can be used in a variety of applications, such as DNA sequencing and detecting DNA fragment in samples, such as for detection of pathogens in samples.
In operation, PCR involves the use of a series of repeated temperature changes or cycles that cause the DNA to melt or denature, yielding two single-stranded DNA molecules that then act as templates. Primers, short DNA fragments, containing sequences complementary to a target region of DNA along with a DNA polymerase, are used to selectively repeat amplification for a particular DNA region or sequence. Typically, two primers are included in a reaction mixture. The primers are single-stranded sequences, but are shorter than the length of the target region of DNA. The primers bind to a complementary part of the DNA strand and the DNA polymerase binds to the primer-DNA hybrid and begins DNA formation of a new DNA strand complementary to the DNA template strand. The process is repeated until multiple copies of the DNA strands have been created.
However, in some instances, the primers can be subject to hetero-dimerization, in which sequences of the primer bind to each other, rather than to the DNA, resulting in short chains of dimers or artifact amplification products, known as primer dimers. These artifact products can form in the early stages of PCR and subsequently be amplified.
An electronic sensor for detection of specific target nucleic acid molecules can include capture probes immobilized on a sensor surface between a set of paired electrodes. An example of a system and method for detecting target nucleic acid molecules is described in U.S. Pat. No. 7,645,574, the entirety of which is herein incorporated by reference. Following PCR, amplified products or amplicons derived from targeted pathogen sequences are captured by the probes via a 5′ single-stranded tail, which was incorporated in the molecules during the amplification process by the use of primers made with an internal replication block. Nano-gold clusters, functionalized with a second capture oligonucleotide having a complementary sequence to a universal 5′-tail tagged onto the other end of the amplified product, are used for localized hybridization to only sensor sites having captured amplification products. Subsequently, using a short treatment with a gold developer reagent, the nano-gold clusters serve as catalytic nucleation sites for metallization, which cascades into the development of a fully conductive film. The presence of the gold film shorts the gap between the electrodes and is measured by a drop in resistance, allowing the presence of the captured amplification products can then be measured. However, primer-only artifact products or possible amplicons derived from spurious nucleic acid molecules, with both primers having the requisite 5′ tails, can react with such sensors in the same way a DNA target would and can result in false positive results.
A method for detecting a nucleic acid molecule in a biological sample includes amplifying a nucleic acid molecule to generate an amplicon having a single 5′-tail and hybridizing the 5′-tail to one of a plurality of capture probes on a surface of a sensor. The amplicon is converted to a single strand molecule and a target-specific catalyst cluster is bound to the single strand molecule. The catalyst cluster is subjected to metallization in order to detect a target nucleic acid.
In an embodiment, a method for detecting a target nucleic acid molecule in a sample with a sensor is disclosed. The sensor includes a first electrode and a second electrode coupled to a sensor surface in a spaced apart arrangement and a plurality of capture probes coupled to the sensor surface between the first electrode and the second electrode. The method includes performing nucleic acid molecule amplification via polymerase chain reaction (PCR) using a first primer having a 5′-tail and a second primer having no 5′-tail to form a plurality of double-stranded amplicons having a first strand with a 5′-tail and a second strand with no tail and hybridizing the plurality of amplicons to the plurality of capture probes. The method further includes converting the plurality of amplicons to a plurality of single strand molecules and binding a catalyst cluster to an interior section of each of the plurality of single strand molecules. In addition, the method includes contacting the plurality of single strand molecules having a catalyst cluster bound thereon with a metal or metal alloy to deposit the metal or metal alloy on the catalyst cluster and determining if an electrical current can be carried between the electrodes. The electrical current between the electrodes indicates the presence of the target nucleic acid molecule in the sample.
In another embodiment, a method for preparing a nucleic acid molecule detector is disclosed. The nucleic acid molecule detector includes a first electrode and a second electrode coupled to a sensor surface in a spaced apart arrangement and a plurality of capture probes coupled to the sensor surface between the first electrode and the second electrode. The method includes receiving a biological sample, amplifying a nucleic acid molecule within the biological sample to generate an amplicon having a single 5′-tail, and binding the 5′-tail of the amplicon to one of the plurality of capture probes. The method additionally includes employing an exonuclease to digest one strand of the amplicon to convert the amplicon to a single strand molecule and synthesizing a target-specific catalyst cluster. The method further includes contacting the catalyst cluster with the single strand molecule of the amplicon to bind the target-specific catalyst cluster to an interior region of the single strand molecule.
An advantage that may be realized in the practice of some disclosed embodiments is reduction or elimination of false positives due to the formation of primer-dimer artifacts or other unintended amplification products.
The above embodiments are exemplary only. Other embodiments are within the scope of the disclosed subject matter.
So that the manner in which the features of the invention can be understood, a detailed description of the invention may be had by reference to certain embodiment, some of which are illustrated in the accompanying drawings. It is to be noted, however, that the drawings illustrate only certain embodiments of this invention and are therefore not to be considered limiting of its scope, for the scope of the disclosed subject matter encompasses other embodiments as well. The drawings are not necessarily to scale, emphasis generally being placed upon illustrating the features of certain embodiments of the invention. In the drawings, like numerals are used to indicate like parts throughout the various views.
At block 26, the hybridized amplicons 30 are converted to single strand molecules 36, as illustrated in
Digestion of the strand 33 exposes the internal sequence region of the tailed strand 31. At block 28 of the method 20 (
Since a generic oligonucleotide is not suitable for binding to internal sequences within the amplicons, the catalyst clusters are target-specific, i.e., the catalyst clusters bind to specific target sequences in the strand 31. Because the primer-dimer artifacts do not include these target sequences, the catalyst clusters 60 do not bind to primer-dimer artifacts, thus avoiding potential false positive measurements. As illustrated in
As illustrated in
Returning to
An existing test developed for plasmodium faliciprium was used to test the method 20 described above.
Because the 5′-tail on the original reverse primer 62 hybridizes to a universal catalyst reagent, modification of the catalyst cluster for use with the method 20 was accomplished by pre-hybridization of the original reverse primer oligonucleotide onto a universal cluster to form target-specific catalyst gold clusters. Preparation of these target-specific catalyst gold clusters included a heated incubation with 1000 fold molar excess of the primer 62, cooling to room temperature, and removal of unbound excess oligonucleotide by washing, repeated twice, via high-speed centrifugation and resuspension with the final reagent buffer. Similarly, a second catalyst reagent 66 with specificity towards a different sequence element located upstream of the first cluster binding sequence was prepared. The ability of this second cluster to effect metallization and detection served as only a preliminary attempt to assess the processivity of the exonuclease in the digestion of sensor-bound amplicons. With this particular derived amplicon, the nuclease must have digested to within at least 95 nucleotides of completely degrading the extraneous DNA strand, leaving a single-stranded tract of about eighty nucleotides available for cluster binding.
Select 5′ to 3′ exonucleases with suitable properties to perform a digestion as per the method 20 were assessed. Two suitable commercially available exonucleases, Lambda and T7, were identified. Lambda was used in this example.
One finding of these experiments was that the Lambda exonuclease could not digest the 5′-tail primer extended strand, which would have caused a loss in the capacity to capture the catalyst reagent. This finding is supported by the longer time coarse digestion experiment illustrated in
The ability of the adaptor modified clusters to hybridize to a more internal site within the 5′-tailed strand were investigated by preparation of the second cluster reagent, described above. This second cluster was formed to bind about thirty nucleotides proximal of the hybridization site of the first cluster. The results of this investigation of illustrated in
Using the method described above for Plasmodium falciparum (P.f.), fifty test cartridge runs were performed. Negative and positive samples consisted respectively of either 10 μL of water or a 1 μL blood culture of P.f. (105) cells diluted in a buffer to 10 μL. After pipetting and sealing a sample into the test cartridge, a fully automated assay, including sample preparation, PCR amplification, and microchip hybridization, nuclease digestion, and metallization reactions, was carried out. Post assay electrical measurements were performed by removal of each microchip board from the cartridge after each test run and visually inspecting each microchip before placing the microchip on a probe station for collection of electrical results. Table 1 presents the raw data from the fifty assays. As indicated by “NO TEST” in the “results” column of Table 1, certain assays were excluded due to machine failure or operator mistakes, as further indicated in the “Notes” column in Table 1. Table 2 indicates the percentages of correct true positives and negatives obtained. As illustrated in Table 2, using a modified, single-tailed amplicons resulted in 100% correct negative measurements and 88% correct positive measurements.
Various combinations of multiplexed reverse transcription PCR (RT-PCR) were assessed using primer sets designed for development of a pan-flavivirus/pan-alphavirus/pan-bunyavirus test. A total of nine primers were used in this assay, with all assayed materials derived from the homogenization, using ultrasonically driven bead-beating, of pooled six to eight mosquitoes that were spiked or non spiked with a virus, VEE-TC83, Dengue, or LaCrosse Virus. Nucleic acid material was isolated from the homogenates using magnetic particle purification, desalted by gel-filtration, and used in the RT-PCR amplification with the appropriate multiplexed primer sets, either pan-flavivirus plus bunyavirus primers or alpha primers, to generate single-stranded 5′-tailed amplicons. The findings indicate that the primer sets for the pan-alpha and pan-flavivirus tests inhibit one another during PCR amplification. To address this problem, the test cartridge provides two separate PCR chambers allowing for interfering primer sets to be run separately and mixed prior to hybridization on the sensor chip.
Possible advantages of the above described method include reduction or elimination of false positive measurements due to primer-dimer artifact formation.
While the present invention has been particularly shown and described with reference to certain exemplary embodiments, it will be understood by one skilled in the art that various changes in detail may be effected therein without departing from the spirit and scope of the invention that can be supported by the written description and drawings. Further, where exemplary embodiments are described with reference to a certain number of elements it will be understood that the exemplary embodiments can be practiced utilizing either less than or more than the certain number of elements.
The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.
To the extent that the claims recite the phrase “at least one of” in reference to a plurality of elements, this recitation is intended to mean at least one or more of the listed elements, and is not limited to at least one of each element. For example, “at least one of an element A, element B, and element C,” is intended to indicate element A alone, or element B alone, or element C alone, or any combination thereof. “At least one of element A, element B, and element C” is not intended to be limited to at least one of an element A, at least one of an element B, and at least one of an element C.
This application claims the benefit of and priority to U.S. Provisional Patent Application Ser. No. 62/337,917, filed May 18, 2016 and entitled “NESTED DETECTION OF PCR PRODUCT,” the entirety of which is incorporated herein by reference.
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/US2017/033255 | 5/18/2017 | WO | 00 |
Number | Date | Country | |
---|---|---|---|
62337917 | May 2016 | US |