Patent Application
20080050724
Embodiments of the amplification and detection method and apparatus are described relative to the several views of the drawings. Where appropriate and only where identical elements are disclosed and shown in more than one drawing, the same reference numeral will be used to represent such identical elements.
Embodiments of the amplification and detection method include a reverse transcription step in which select gene sequences are reverse transcribed, thereby reducing if not eliminating the reverse transcription of non-specific products, that is gene sequences that are not to be detected. The reverse transcribed gene-specific sequences are then amplified and detected. The amplification and detection method can be simultaneously applied to multiple different gene-specific sequences. For simplicity, the amplification and detection method is described in terms of reverse transcribing gene-specific RNA sequences into corresponding gene-specific cDNA sequences, and then amplifying the gene-specific cDNA sequences. It is understood that the amplification and detection method is applicable to any type of gene sequence.
At the step 100, a sample is provided that includes a low copy RNA strand. An objective is to detect if a specific gene sequence in the RNA is present within the sample. At the step 105 a pair of gene-specific primers are added to the sample. By way of example, the specific gene sequence that is to be targeted and amplified is referred to as gene 1. In this case, the pair of gene-specific primers are specific to gene 1. One of the pair of gene-specific primers, referred to in this case as the gene 1 reverse transcription (RT) primer, is configured to bond with the RNA strand at one end of the gene. At the step 110, reagents are added to the sample. It is understood that the step 105 and the step 110 can be combined as a single step. The amplification and detection method combines a reverse transcription step and a first amplification step within a single processing chamber. The pair of gene-specific primers including the gene 1 RT primer, reagents for the reverse transcription step, and reagents for the first amplification step are all added to the original sample prior to performing the reverse transcription step. At the step 115, the mixture including the sample, the pair of gene-specific primers, and the reagents, is incubated at a first temperature for a first period of time. During this incubation period, the gene 1 RT primer binds to an RNA strand if the RNA strand includes the gene 1. The bound gene 1 RT primer initiates the reverse transcription of the gene 1 in the RNA to a corresponding cDNA sequence, including the gene 1. During this incubation step, the gene-specific sequence in the RNA is identified and reverse transcribed into the corresponding specific cDNA sequence.
After the first period of time, a first amplification step is performed on the reverse transcribed sample solution at the step 120. The first amplification step is performed by thermal cycling, or PCR. In the first amplification and detection method, the first amplification step is limited to the linear phase. Linear phase amplification is maintained during about 5-15 thermal cycles. In some embodiments, the incubation step in which reverse transcription takes place and the first amplification step are performed in a single processing chamber. Since the pair of gene-specific primers and the reagents needed for reverse transcription and the first amplification are previously added, there is no need to add reagents and/or additional primers to the sample solution in between the incubation step and the first amplification step.
At the step 125, additional reagents are added to the first amplified sample solution, thereby diluting the first amplified sample solution. In some embodiments, an additional dilution step is performed whereby an additional neutral solution, such as water, is added in addition to the reagents added in the step 125. The reagents include detection chemistries, such as TaqMan® probes. At the step 130, a pair of gene-specific primers targeted to the gene 1 is added to the first amplified sample solution. The pair of gene-specific primers added at the step 130 can be the same as the pair of gene-specific primers added at the step 105. Alternatively, the pair of gene-specific primers added at the step 130 are not the same as the pair of gene-specific primers added at the step 105. Instead, each of the pair of gene-specific primers added in the step 130 are internal to the pair of gene-specific primers added at the step 105. In general, the pair of gene-specific primers added at the step 130 is equal to or internal to the pair of gene-specific primers added at the step 105. At the step 135, a second amplification step is performed on the first amplified sample solution. As with the first amplification step, the second amplification step is performed by thermal cycling. In some embodiments, up to 40 thermal cycles are performed. Alternatively, more than 40 thermal cycles are performed. In general, the number of thermal cycles performed during the second amplification step is sufficient for entering into an exponential phase amplification and reaching saturation. Saturation is the point where additional cycles do not increase the yield of specific product. The result is a sample solution including an amplified number of cDNA strands including the gene-specific sequence. At the step 140, the amplified specific gene sequence is detected, if present. Typically, during the second amplification step the detection chemistries are stimulated, such as releasing a detectable flourescent probe, which are then detected using any number of conventional detection means.
The first amplification and detection method is extendable so that it is applied to the amplification and detection of multiple different types of specific low copy targets.
At the step 160, reagents used for reverse transcription and a first amplification step are added to the sample. It is understood that the step 155 and the step 160 can be combined as a single step. At the step 165, the mixture including the sample, the multiple different pairs of gene-specific primers, and the reagents, is incubated at a set temperature for a period of time. During this incubation period, a specific gene-specific primer bonds to an RNA strand if the RNA strand includes the specific gene sequence corresponding to the specific gene-specific primer. The bound gene-specific primer initiates the reverse transcription of the specific gene sequence in the RNA to a corresponding cDNA strand including the specific gene sequence. During this incubation step, each specific gene sequence present is identified and reverse transcribed into the corresponding specific gene sequence in the cDNA.
After the period of time, a first amplification step is performed on the reverse transcribed sample solution at the step 170. The first amplification step is performed by thermal cycling, or PCR. In the second amplification and detection method, the first amplification step is limited to the linear phase. Linear phase amplification is maintained during about 5-15 thermal cycles. In some embodiments, the incubation step in which reverse transcription takes place and the first amplification step are performed in a single processing chamber. Since the multiple pairs of gene-specific primers and the reagents needed for reverse transcription and the first amplification step are previously added, there is no need to add reagents and/or additional gene-specific primer pairs to the sample solution in between the incubation step and the first amplification step.
At the step 175, the first amplified sample solution is divided into sample portions. In some embodiments, the number of sample portions is equal to the number of targets (gene-specific sequences) to be detected. For example, where the amplification and detection method is configured to detect the presence of 20 different targets, the first amplified sample solution is divided into 20 sample portions. Alternatively, the sample solution is divided into fewer than the number of detected targets. At the step 180, additional reagents are added to each sample portion. In some embodiments, additional dilution can be achieved by adding a neutral solution, such as water, to one or more of the sample portions. The reagents include detection chemistries, such as TaqMan® probes. At the step 185, one or more different pairs of gene-specific primers are added to each sample portion. Each pair of gene-specific primers is targeted to a specific gene sequence. The total number of different pairs of gene-specific primers is equal to the number of targets to be detected. Each different pair of gene-specific primers added at the step 185 corresponds to the same specific gene sequence as the pair of gene-specific primers added at the step 155. For example, where the specific gene sequence is gene 2, the pair of gene-specific primers added at the step 155 and the pair of gene-specific primers added at the step 185 each target the gene 2. Each pair of gene-specific primers added at the step 185 can be the same as the corresponding pair of gene-specific primers added at the step 155. Alternatively, each pair of gene-specific primers added at the step 185 is internal to the corresponding pair of gene-specific primers added at the step 155. In the case where the number of sample portions equals the number of detected targets, one different pair of gene-specific primers is added to each sample portion. In the case where the sample solution is divided into fewer than the number of detected targets, then the different pairs of gene-specific primers are divided into groups, and one group of primer pairs is added to each sample portion.
At the step 190, a second amplification step is performed on each sample portion. As with the first amplification step, the second amplification step is performed by thermal cycling. In some embodiments, up to 40 thermal cycles are performed. Alternatively, more than 40 thermal cycles are performed. In general, the number of thermal cycles performed during the second amplification step is sufficient for entering into an exponential phase amplification and reaching saturation. The result is a sample portion including an amplified number of specific gene sequences corresponding to the specific gene-specific primer pairs added to the sample portion at the step 185. At the step 195, the amplified specific gene sequences are detected, if present, in each sample portion. Typically, during the second amplification step the detection chemistries are stimulated, such as releasing a detectable flourescent probe, which are then detected using any number of conventional detection means.
At the step 300, a sample is provided that includes a low copy RNA strand. An objective is to detect if a specific gene sequence in the RNA is present within the sample. At the step 305 a pair of gene-specific primers are added to the sample. By way of example, the specific gene sequence that is to be targeted and amplified is referred to as gene 1. In this case, the pair of gene-specific primers are specific to gene 1. One of the pair of gene-specific primers, referred to as the gene I reverse transcription (RT) primer, is configured to bond with the RNA strand at one end of the gene. At the step 310, reagents are added to the sample. It is understood that the step 305 and the step 310 can be combined as a single step. The third amplification and detection method combines a reverse transcription step and a first amplification step within a single processing chamber. The pair of gene-specific primers including the gene 1 RT primer, reagents for the reverse transcription step, and reagents for the first amplification step are all added to the original sample prior to performing the reverse transcription step. At the step 315, the mixture including the sample, the pair of gene-specific primers, and the reagents, is incubated at a first temperature for a first period of time. During this incubation period, the gene 1 RT primer binds to an RNA strand if the RNA strand includes the gene 1. The bound gene 1 RT primer initiates the reverse transcription of the gene 1 in the RNA to a corresponding cDNA sequence, including the gene 1. During this incubation step, the gene-specific sequence in the RNA is identified and reverse transcribed into the corresponding specific cDNA sequence.
After the first period of time, a first amplification step is performed on the reverse transcribed sample solution at the step 320. The first amplification step is performed by thermal cycling, or PCR. The number of thermal cycles performed during the first amplification step is sufficient to move beyond the linear phase of amplification and into the exponential phase. However, the number of thermal cycles performed during the first amplification stage is less than the number of thermal cycles sufficient to reach saturation. Saturation is the point where additional cycles do not increase the yield of specific product. For example, the first amplification step is performed for 20-25 cycles. In some embodiments, the incubation step in which reverse transcription takes place and the first amplification step are performed in a single processing chamber. Since the pair of gene-specific primers and the reagents needed for reverse transcription and amplification are previously added, there is no need to add reagents and/or additional primers to the sample solution in between the incubation step and the first amplification step.
At the step 325, additional reagents are added to the first amplified sample solution, thereby diluting the first amplified sample solution. In some embodiments, an additional dilution step is performed whereby an additional neutral solution, such as water, is added in addition to the reagents added in the step 325. The reagents include detection chemistries, such as TaqMan® probes. At the step 330, a pair of gene-specific primers targeted to the gene I is added to the first amplified sample solution. The pair of gene-specific primers added at the step 330 can be the same as the pair of gene-specific primers added at the step 305. Alternatively, the pair of gene-specific primers added at the step 330 are not the same as the pair of gene-specific primers added at the step 305. Instead, each of the pair of gene-specific primers added in the step 330 are internal to the pair of gene-specific primers added at the step 305. In general, the pair of gene-specific primers added at the step 330 is equal to or internal to the pair of gene-specific primers added at the step 305.
At the step 335, a second amplification step is performed on the first amplified sample solution. As with first amplification step, the second amplification step is performed by thermal cycling. In some embodiments, 20-25 thermal cycles are performed. Alternatively, more or less than 20-25 thermal cycles are performed. In general, the number of thermal cycles performed during the second amplification step is a minimum number sufficient to generate a detectable level of the copy target (e.g. each gene-specific sequence being targeted). In most applications, this minimum number of thermal cycles is insufficient to reach saturation. An advantage of the third amplification and detection method is the elimination of non-specific product generated during the second amplification step. The result is a sample solution including an amplified number of cDNA strands including the gene-specific sequence. At the step 340, the amplified specific gene sequence is detected, if present. Typically, during the second amplification step the detection chemistries are stimulated, such as releasing a detectable flourescent probe, which are then detected using any number of conventional detection means.
At the step 350, a sample is provided that includes multiple low copy RNA strands. An objective is to detect if multiple different specific gene sequences are present within the sample. At the step 355 multiple different pairs of gene-specific primers are added to the sample. At the step 360, reagents used for reverse transcription and amplification are added to the sample. It is understood that the step 355 and the step 360 can be combined as a single step. At the step 365, the mixture including the sample, the multiple different pairs of gene-specific primers, and the reagents, is incubated at a set temperature for a period of time. During this incubation period, a specific gene-specific primer bonds to an RNA strand if the RNA strand includes the specific gene sequence corresponding to the specific gene-specific primer. The bound gene-specific primer initiates the reverse transcription of the specific gene sequence in the RNA to a corresponding cDNA strand including the specific gene sequence. During this incubation step, each specific gene sequence present is identified and reverse transcribed into the corresponding specific gene sequence in the cDNA.
After the period of time, a first amplification step is performed on the reverse transcribed sample solution at the step 370. The first amplification step is performed by thermal cycling, or PCR. The number of thermal cycles performed during the first amplification step is sufficient to move beyond the linear phase of amplification and into the exponential phase. However, the number of thermal cycles performed during the first amplification stage is less than the number of thermal cycles sufficient to reach saturation. For example, the first amplification step is performed for 20-25 cycles. In some embodiments, the incubation step in which reverse transcription takes place and the first amplification step are performed in a single processing chamber. Since the multiple pairs of gene-specific primers and the reagents needed for reverse transcription and the first amplification step are previously added, there is no need to add reagents and/or additional gene-specific primer pairs to the sample solution in between the incubation step and the first amplification step.
At the step 375, the first amplified sample solution is divided into sample portions. In some embodiments, the number of sample portions is equal to the number of targets (gene-specific sequences) to be detected. Alternatively, the sample solution is divided into fewer than the number of detected targets. At the step 380, additional reagents are added to each sample portion. In some embodiments, additional dilution can be achieved by adding a neutral solution, such as water, to one or more of the sample portions. The reagents include detection chemistries, such as TaqMan® probes. At the step 385, one or more different pairs of gene-specific primers are added to each sample portion. Each pair of gene-specific primers is targeted to a specific gene sequence. The total number of different pairs of gene-specific primers is equal to the number of targets to be detected. Each different pair of gene-specific primers added at the step 385 corresponds to the same specific gene sequence as the pair of gene-specific primers added at the step 355. For example, where the specific gene sequence is gene 2, the pair of gene-specific primers added at the step 355 and the pair of gene-specific primers added at the step 385 each target the gene 2. Each pair of gene-specific primers added at the step 385 can be the same as the corresponding pair of gene-specific primers added at the step 355. Alternatively, each pair of gene-specific primers added at the step 385 is internal to the corresponding pair of gene-specific primers added at the step 355. In the case where the number of sample portions equals the number of detected targets, one different pair of gene-specific primers is added to each sample portion. In the case where the sample solution is divided into fewer than the number of detected targets, then the different pairs of gene-specific primers are divided into groups, and one group of primer pairs is added to each sample portion.
At the step 390, a second amplification step is performed on each sample portion. As with the first amplification step, the second amplification step is performed by thermal cycling. In some embodiments, 20-25 thermal cycles are performed during the second amplification step. Alternatively, more or less than 20-25 thermal cycles are performed. In general, the number of thermal cycles performed during the second amplification step is a minimum number sufficient to generate a detectable level of the copy target (e.g. each specific gene sequence being targeted). In most applications, this minimum number of thermal cycles is insufficient to reach saturation. An advantage of the fourth amplification and detection method is the elimination of non-specific product generated during the second amplification step. The result is a sample portion including an amplified number of specific gene sequences corresponding to the specific gene-specific primer pairs added to the sample portion at the step 385. At the step 395, the amplified specific gene sequences are detected, if present, in each sample portion.
Typically, during the second amplification step the detection chemistries are stimulated, such as releasing a detectable flourescent probe, which are then detected using any number of conventional detection means.
In some embodiments, the amplification and detection method is implemented within an amplification and detection apparatus.
The amplification apparatus 200 includes a collection vessel 205, an incubation and first amplification chamber 210, a second amplification chamber 215, and a mixing solutions module 225 coupled together via microfluidic circuitry. The amplification apparatus 200 also includes a controller 220 coupled to control the operation of the microfluidic circuitry, the collection vessel 205, the incubation and first amplification chamber 210, the second amplification chamber 215, and the mixing solutions module 225. The controller 220 also controls fluid flow into and out of the amplification apparatus 200. In some embodiments, the controller 220 is also coupled to control the operation of the detection apparatus 230.
The collection vessel 205 is configured to receive the input sample. The amplification and detection apparatus is configured to amplify and detect the presence of one or more low copy targets that might be included in the input sample. The sample is then directed to the incubation and first amplification chamber 210. The incubation and first amplification chamber 210 is configured to perform the incubation and first amplification steps described above. The mixing solutions module 225 is coupled to the incubation and first amplification chamber 210 to provide the one or more different pairs of gene-specific primers and the reagents necessary to perform the incubation and first amplification steps. The mixing solutions module 225 includes multiple containment vessels for storing the gene-specific primers and the reagents. The various primers and reagents can be stored independently or in any pre-mixed combination. In some embodiments, the incubation and first amplification chamber 210 is the collection vessel 205, and the input sample is provided to the incubation and first amplification chamber 210.
The incubation and first amplification chamber 210 outputs the first amplified sample solution to the second amplification chamber 215. The second amplification chamber 215 is configured to perform the second amplification step described above. Where the amplification and detection method is configured to divide the first amplified sample solution into sample portions, the microfluidic circuitry is configured to divide the first amplified sample solution and to provide each sample portion to the second amplification chamber 215. In this case, the second amplification chamber 215 includes multiple separate amplification chambers, each to receive a sample portion. Alternatively, the amplification and detection apparatus includes a metering and distribution module that is configured to divide the first amplified sample solution into the sample portions.
The mixing solutions module 225 is coupled to the second amplification chamber 215 to provide the one or more different pairs of gene-specific primers and the reagents necessary to perform the second amplification step. The second amplification chamber 215 outputs the second amplified sample solution, or each second amplified sample portion, to the detection apparatus 230. The detection apparatus 230 is configured to detect if one or more specific targets are present within the second amplified sample solution, or within each of the second amplified sample portions.
In some embodiments, the amplification and detection apparatus is configured to automatically perform one, some, or all of the steps of the amplification and detection method described above. In this manner, one, some, or all of the steps of the amplification and detection method can be automated.
The amplification and detection apparatus can be implemented as a stand-alone device or as part of a larger system, such as a particle collection and detection system described in the co-owned and co-pending U.S. Patent Application Serial No. (MFSI-00700), entitled “An Integrated Substance Collection and Detection System,” which is hereby incorporated by reference.
The amplification and detection method includes a two-step RT-PCR approach for detection of multiple low-abundance targets. This method requires significantly less starting material than conventional approaches. Such improved sensitivity allows detection in small samples, such as 1-100 cells. In some embodiments, detection of one or more low copy targets is achieved via real-time PCR using “RT-amplification” product generated by controlled hot start multiplex RT-PCR. In contrast with conventional approaches, such as TaqMan®, this method requires lower amounts of starting material and therefore can be applied to detect multiple low expressed targets in environmental samples. The amplification and detection method allows simultaneous quantification of hundreds of targets using as little as 2.5 fg of sample, whereas conventional assays require substantially larger amounts of nucleic acid, usually from 10 ng to 1 ug per reaction. Conventional gene microarrays require even larger amounts of starting RNA (1-10 ug), and individual gene probes in labeled complex cDNA/cRNA mixtures may have unique secondary structures, melting temperatures, and reassociation rates which makes hybridization of all gene probes under optimum condition nearly impossible. At this time, no other technique offers the potential for simultaneous rapid, accurate, and reliable quantification of multiple targets in small samples (1-100 cells).
The amplification and detection methods are described above as performing an amplification method on a sample that includes one or more different RNA sequences. In some embodiments, the initial sample also includes one or more different DNA sequences. In other embodiments, the sample may not include any RNA sequences, but instead include only one or more different DNA sequences. In such cases, any DNA sequences present in the sample during the reverse transcription step remain unchanged. For example, if a sample includes both RNA sequences and DNA sequences, or just DNA sequences, when the incubation step is performed whereby gene-specific RNA sequences are reverse transcribed into corresponding cDNA sequences, this process has no impact on the DNA sequences included within the sample. In the case where both RNA sequences and DNA sequences are present within the sample, the gene-specific RNA sequences are reverse transcribed, while the DNA sequences remain unaffected. In the case where only DNA sequences are present in the sample, the incubation step does not yield any results, since no RNA sequences are present. In either case, this eliminates the need to first determine if the sample includes RNA sequences, DNA sequences, or both, and then subsequently having to alter the process according to that knowledge. During the first amplification step and the second amplification step, any original DNA sequences that correspond to the gene-specific primers are amplified, similar to any gene-specific reverse transcribed cDNA sequences as described above.
The present invention has been described in terms of specific embodiments incorporating details to facilitate the understanding of the principles of construction and operation of the invention. Such reference herein to specific embodiments and details thereof is not intended to limit the scope of the claims appended hereto. It will be apparent to those skilled in the art that modifications may be made in the embodiment chosen for illustration without departing from the spirit and scope of the invention.
