Abstract:
The present invention relates to systems and methods for monitoring the amplification of DNA molecules and the dissociation behavior of the DNA molecules.

Description:
This application is a continuation of and claims priority to U.S. patent application Ser. No. 12/552,645, filed on Sep. 2, 2009, now issued U.S. Pat. No. 8,306,294, which is a continuation of and claims priority to U.S. patent application Ser. No. 11/947,237, filed on Nov. 29, 2007, now U.S. Pat. No. 7,593,560, which claims the benefit of U.S. Provisional Patent Application Ser. No. 60/861,712, filed on Nov. 30, 2006, each of which are incorporated herein by reference in their entirety. 
    
    
     BACKGROUND 
     1. Field of the invention 
     The present invention relates to systems and methods for monitoring the amplification of DNA molecules and the dissociation behavior of the DNA molecules. 
     2. Discussion of the Background 
     The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. Polymerase chain reaction (PCR) is a well-known technique for amplifying DNA. 
     With PCR, one can quickly produce millions of copies of DNA starting from a single template DNA molecule. PCR includes a three phase temperature cycle of denaturation of the DNA into single strands, annealing of primers to the denatured strands, and extension of the primers by a thermostable DNA polymerase enzyme. This cycle is repeated a number of times so that at the end of the process there are enough copies to be detected and analyzed. For general details concerning PCR, see Sambrook and Russell,  Molecular Cloning—A Laboratory Manual  (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000);  Current Protocols in Molecular Biology , F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley &amp; Sons, Inc., (supplemented through 2005) and  PCR Protocols A Guide to Methods and Applications , M. A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990). 
     In some applications, it is important to monitor the accumulation of DNA products as the amplification process progresses. Real-time PCR refers to a growing set of techniques in which one measures the buildup of amplified DNA products as the reaction progresses, typically once per PCR cycle. Monitoring the amplification process over time allows one to determine the efficiency of the process, as well as estimate the initial concentration of DNA template molecules. For general details concerning real-time PCR see  Real - Time PCR: An Essential Guide , K. Edwards et al., eds., Horizon Bioscience, Norwich, U.K. (2004). 
     More recently, a number of high throughput approaches to performing PCR and other amplification reactions have been developed, e.g., involving amplification reactions in microfluidic devices, as well as methods for detecting and analyzing amplified nucleic acids in or on the devices. Thermal cycling of the sample for amplification is usually accomplished in one of two methods. In the first method, the sample solution is loaded into the device and the temperature is cycled in time, much like a conventional PCR instrument. In the second method, the sample solution is pumped continuously through spatially varying temperature zones. See, for example, Lagally et al. ( Anal Chem  73:565-570 (2001)), Kopp et al. ( Science  280:1046-1048 (1998)), Park et al. ( Anal Chem  75:6029-6033 (2003)), Hahn et al. (WO 2005/075683), Enzelberger et al. (U.S. Pat. No. 6,960,437) and Knapp et al. (U.S. Patent Application Publication No. 2005/0042639). 
     Once there are a sufficient number of copies of the original DNA molecule, the DNA can be characterized. One method of characterizing the DNA is to examine the DNA&#39;s dissociation behavior as the DNA transitions from double stranded DNA (dsDNA) to single stranded DNA (ssDNA) with increasing temperature. The process of causing DNA to transition from dsDNA to ssDNA is sometimes referred to as a “high-resolution temperature (thermal) melt (HRTm)” process, or simply a “high-resolution melt” process. 
     Accordingly, what is desired is a system for monitoring the DNA amplification process and for determining the DNA&#39;s dissociation behavior. 
     SUMMARY OF THE INVENTION 
     The present invention relates to systems and methods for performing and monitoring real-time PCR and HRTm analysis. 
     In one aspect, the present invention provides a method that includes the steps of: introducing a sample of a solution comprising nucleic acid into a microchannel; forcing the sample to move though the channel; defining a first window of a pixel array of an image sensor; defining a second window of the pixel array, wherein the center of the second window is spaced apart from the center of the first window; and while the sample is moving through the microchannel, performing the steps of: (a) exposing the first window of the pixel array to light emitted from the sample at a time when the sample is within a field of view of first window and then selectively outputting first image data from the pixel array, wherein the step of selectively outputting the first image data from the pixel array comprises outputting data from only the first window of the pixel array; and (b) after performing step (a), exposing the second window of the pixel array to light emitted from the sample at a time when the sample is within a field of view of second window and then selectively outputting second image data from the pixel array, wherein the step of selectively outputting the second image data from the pixel array comprises outputting data from only the second window of the pixel array. The step of defining the second window may occur after step (a). 
     In some embodiments, when the step of exposing the first window of the pixel array to light emitted from the sample is performed, the center of the first window corresponds substantially to the center of the sample, and when the step of exposing the second window of the pixel array to light emitted from the sample is performed, the center of the second window corresponds substantially to the center of the sample. 
     In some embodiments the size of the second window may be less than or greater than the size of the first window, and the step of defining the second window includes processing the first image data to determine whether the amount of light received at a pixel located at an edge of the first window exceeds or equals a predetermined threshold. 
     In some embodiments, the method may also include the steps of: receiving from a first sensor a first signal indicating that the sample has been detected by the first sensor and receiving from a second sensor a second signal indicating that the sample has been detected by the second sensor, wherein the first sensor is positioned to detect when the sample enters the field of view of the image sensor and the second sensor is positioned to detect when the sample leaves the field of view of the image sensor. 
     In some embodiments, the step of defining the first window of the pixel array comprises determining the size of the window, wherein the determination is based, at least in part, on the length of the sample, and the step of defining the second window of the pixel array comprises determining the location of the center of the second window, wherein the determination is based, at least in part, on a speed at which the sample moves through the channel. 
     In another aspect, the present invention provides a system that includes the following elements: a chip comprising a microchannel for receiving a sample of solution comprising nucleic acid and for providing a path for the sample to traverse; an image sensor having a pixel array, wherein at least a portion of the microchannel is within a field of view of the pixel array; and an image sensor controller configured to: (a) read only a first window of the pixel array at time when the sample is within a field of view of the first window, and (b) read only a second window of the pixel array at time when the sample is within a field of view of the second window, wherein the center of the second window is spaced apart from the center of the first window. 
     In another aspect, the present invention provides a method that includes the following steps: introducing a sample of a solution comprising nucleic acid into a channel; causing the sample to move though the channel; defining a first window of a pixel array of an image sensor; defining a second window of the pixel array, wherein the center of the second window is spaced apart from the center of the first window; while the sample is moving through the microchannel, performing the steps of: (a) windowing the image sensor so that image data from the first window is output to a data buffer, wherein said image data comprises data from which the intensity of emissions from the sample can be determined; and (b) after performing step (a), windowing the image sensor so that image data from the second window is output to a data buffer, wherein said image data comprises data from which the intensity of emissions from the sample can be determined. 
     In yet another aspect, the present invention provides a method that includes the following steps: introducing a first sample of a solution comprising nucleic acid into a first channel; introducing a second sample of a solution comprising nucleic acid into a second channel; causing the first sample to move though the first channel; causing the second sample to move though the second channel; defining a first window of a pixel array of an image sensor, wherein at least a portion of the first channel is within the field of view of the first window; defining a second window of the pixel array, wherein at least a portion of the second channel is within the field of view of the second window and wherein the center of the second window is spaced apart from the center of the first window; while the samples are moving through the respective channels, performing the steps of: (a) windowing the image sensor so that image data from the first window is output to a data buffer, wherein said image data comprises data from which the intensity of emissions from the first sample can be determined; and (b) after performing step (a), windowing the image sensor so that image data from the second window is output to a data buffer, wherein said image data comprises data from which the intensity of emissions from the second sample can be determined. 
     The above and other embodiments of the present invention are described below with reference to the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The accompanying drawings, which are incorporated herein and form part of the specification, illustrate various embodiments of the present invention. In the drawings, like reference numbers indicate identical or functionally similar elements. Additionally, the left-most digit(s) of a reference number identifies the drawing in which the reference number first appears. 
         FIG. 1  illustrates a nucleic acid analysis system  100  according to an embodiment. 
         FIG. 2  is a top view of biochip  102  according to some embodiments. 
         FIG. 3  is a functional block diagram illustrating an embodiment of image processing system  112 . 
         FIG. 4  shows an exemplary pixel array  400 . 
         FIG. 5  illustrates a process according to an embodiment. 
         FIG. 6  pictorially illustrates some of the steps of the process shown in  FIG. 5 . 
         FIG. 7  shows a first window of a pixel array and a second window of the pixel array. 
         FIG. 8  pictorially illustrates some of the steps of the process shown in  FIG. 5 . 
         FIG. 9  pictorially illustrates a process according to an embodiment. 
         FIG. 10  pictorially illustrates a process according to an embodiment. 
         FIG. 11  is a flow chart illustrating a process according to another embodiment. 
         FIG. 12  pictorially illustrates some of the steps of the process shown in  FIG. 11 . 
     
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     Referring to the drawings,  FIG. 1  illustrates a nucleic acid analysis system  100  according to an embodiment. As shown in  FIG. 1 , system  100  includes a microfluidic biochip  102 .  FIG. 2  is a top view of biochip  102  according to some embodiments. As shown in  FIG. 2 , biochip  102  includes a number of microfluidic channels  202 . In the example shown, there are 4 microfluidic channels (i.e., channels  202   a,b,c,d ), but it is contemplated that chip  102  may have more or less than 4 channels. 
     In some embodiments, when system  100  is in use, at least one channel  202  receives a sample (or “bolus”) of a solution containing real-time PCR reagents. A force may be used to cause the bolus to travel through the channel  202  and a thermal generating apparatus  114  may be used to cycle the temperature of the bolus as described above while the bolus moves through the channel  202 . One system and method for performing PCR in a microfluidic device is disclosed in U.S. patent application Ser. No. 11/505,358, filed on Aug. 17, 2006, incorporated herein by reference. 
     As further shown in  FIG. 1 , analysis system  100  may further include an image sensor  108 , a controller  110  configured to control image sensor  108 , and an image processing system  112  configured to process the image data produced by image sensor  108 . Image sensor  108  may be implemented using a CMOS image sensor, a CCD image sensor, or other image sensor. For example, in one embodiment, sensor  108  is a CMOS sensor with an effective 12.7 mega pixel resolution and having a size of 36×24 mm, which is available from Canon Inc. 
     Referring now to  FIG. 3 ,  FIG. 3  is a functional block diagram illustrating an embodiment of image processing system  112 . As shown in  FIG. 3 , system  112  receives data output from image sensor  108 . System  112  may include an amplifier  302  to amplify the data from image sensor  108 . In one nonlimiting embodiment, amplifier  302  may amplify the data for greater sensitivity. The amplified data may be converted to a digital signal by, for example, a 16 bit analog-to-digital (A/D) converter  304 . In one embodiment, utilization of a 16 bit AID converter provides a high level of dynamic range and low end bit resolution. The digital signal output from AID converter  304  may be processed by a framing circuit  306 , which may be configured to store data produced during a PCR process in a zone  1  data buffer  303  and store data produced during a HRTm process in a zone  2  data buffer  310 . A programmable data processor  312  may be programmed to process data in buffers  310  and  303  to, among other things, determine and record the intensity of the fluorescence from samples that undergo the PCR and HRTm processing. 
     As is well known in the art of imaging, image sensor  108  may have an array of pixels. Referring now to  FIG. 4 ,  FIG. 4  shows an exemplary pixel array  400 . For the sake of clarity, pixel array  400  includes only 400 pixels. However, it is well understood that the pixel array of image sensor  108  may have over 1 million pixels. In at least one embodiment, image sensor  108 , lens  140  and chip  102  are arranged so that at least a significant portion of each channel of chip  102  is within the field of view of the pixel array  400  of image sensor  108 . Also, in at least one exemplary embodiment the image sensor  108  has the ability to read out a predefined portion or “window” of the pixel array (this is known as windowing).  FIG. 4  shows an example window  402 , which consists of pixels  433 ,  434 ,  443  and  444 . As is well known in the art, image sensor  108  may have the ability to read out only the pixels that make up window  402  (i.e., to obtain image data from only those pixels within window  402 ). For example, image sensor  108  may have pixel-row and pixel-column select circuits that enable one to read out only a particular window. Embodiments of the present invention can make use of this feature as described below. 
     Referring now to  FIG. 5 ,  FIG. 5  is a flow chart illustrating a process  500  according to an embodiment of the invention. Process  500  may begin in step  502 , where a first sample of a solution comprising nucleic acid is introduced into a channel of chip  102  (for the sake of discussion we will assume the sample is introduced into channel  202   a ). In step  504 , a second sample of a solution comprising nucleic acid is introduced into another channel  202  of chip  102  (for the sake of discussion we will assume the sample is introduced into channel  202   b ). Steps  502  and  504  are illustrated in  FIG. 6 , which shows the first sample (i.e., sample  601 ) in channel  202   a  and shows the second sample (i.e., sample  602  in channel  202   b ). In step  506 , a pressure force is applied to samples  601  and  602  causing them to move through channels  202   a,b , respectively. 
     In step  508 , a first window of pixel array  400  is defined such that at least a portion of channel  202   a  is within the field of view of the first window. In step  510 , a second window of pixel array  400  is defined such that at least a portion of channel  202   b  is within the field of view of the second window and such that the center of the second window is spaced apart from the center of the first window. Steps  508  and  510  are illustrated in  FIG. 7 , which shows a first window  701  of pixel array  400  and a second window  702  of pixel array  400 . 
     While sample  601  moves through the field of view of window  701 , step  512  may be performed. Similarly, while sample  601  moves through the field of view of window  701 , step  520  may be performed. In step  512  the temperature of sample  601  is cycled a number of times to achieve amplification of the nucleic acid present within sample  601  and in step  520  the temperature of sample  602  is cycled to achieve amplification of the nucleic acid present within sample  602 . 
     While steps  512  and  520  are being performed, steps  514  and  522  are performed. In step  514 , controller  110  windows image sensor  108  so that image data from window  701  is output to a data buffer and in step  516  the image data is processed by image processing system  112 . This image data comprises data from which the intensity of emissions from sample  601  can be determined because when step  514  is performed, sample  601  is within the field of view of window  701 , as illustrated in  FIG. 8 . Similarly, in step  522 , controller  110  windows image sensor  108  so that image data from window  702  is output to a data buffer and in step  524  the image data is processed by image processing system  112 . This image data comprises data from which the intensity of emissions from sample  602  can be determined because when step  522  is performed, sample  602  is within the field of view of window  702 , as illustrated in  FIG. 8 . As shown in  FIG. 8 , the width of windows  701  and  702  may be configured to be slightly greater than the width of the respective channels (e.g., the width of window  701  may be equal to the pixel width of channel  202   a  plus not more than several pixels). 
     As illustrated in  FIG. 5 , steps  514 ,  516 ,  522  and  524  may be repeated. 
     In some embodiments, prior to performing steps  514  and  522  again, windows  701  and  702  may be redefined. For example, windows  701  and/or  702  may be made smaller so that less image data is transferred to the data buffers on subsequent performance of step  514  and/or  522 . In some embodiments, the window may be redefined so that the size of the window is equal to the pixel size of the sample plus a few pixels, and the center of the window corresponds substantially to the location of the center of the sample. 
     To determine the pixel size of the sample, image processing system  112  may be programmed to determine the pixels of pixel array that received at least a predetermined threshold of light from the sample. The window may then be defined to include those pixels plus, for each pixel, not more than a predetermined number of neighboring pixels (e.g., not more than about 5 neighboring pixels). This process is illustrated in  FIG. 9 . As shown in  FIG. 9 , the shaded pixels represent the pixels that received at least a predetermined amount of light from the sample during a predetermined integration period. Given this information, a window can be defined to include not only these pixels, but also neighboring pixels for each pixel. Such a window  990  is shown in  FIG. 9 . As illustrated, window  990  includes not only not only the pixels that received at least the predetermined amount of light, but also two neighboring pixels for each said pixel. 
     In one embodiment, to determine the point of the pixel array  400  that corresponds to the location of the center of the sample  601  at some specific point in time, processing system  112  may compute the location based on knowledge of the location of the center of the sample  601  at some previous point in time (e.g., the point in time when step  514  was last performed), the average velocity of the sample during the time period between the specific point in time and the previous point in time, and the time difference between the specific point in time and the previous point in time. The location of the center of the sample  601  at some previous point in time and the average velocity of the sample may be known or may be derived from image data captured by image sensor  108 . This process is illustrated in  FIG. 10 . 
       FIG. 10  shows a first group of pixels that received at least a predetermined amount of light from the sample at time t 1 . Pixel  1001  is in the center of this group of pixels. With this information, the point of pixel array  400  that corresponds to the location of the center of the sample at time t 2  can be determined using the following formula: S*(t 2 −t 1 ), where s is the speed of the sample as it moves through the channel in units of pixels/unit of time. For example if t 2 −t 1  equals 1 second and s equals 5 pixels per second and one knows the sample moves in the direction of arrow  1090 , then one can determine that at time t 2  the center of the sample will be at pixel  1099 . 
     Referring now to  FIG. 11 ,  FIG. 11  is a flow chart illustrating a process  1100  according to an embodiment of the invention. Process  1100  may begin in step  1102 , where a sample of a solution comprising nucleic acid is introduced into a channel of chip  102 . In step  1104 , a force is applied to the sample causing it to move through the channel. 
     While the sample is within at least some portion of the channel, step  1110  is performed. In step  1110  the temperature of the sample is cycled a number of times to achieve amplification of the nucleic acid present within sample. 
     While step  1110  is being performed, the following steps are performed. In step  1111  a window of pixel array  400  is defined such that at some particular point in time the sample will be in the field of view of the window. Preferably, the window is sized and positioned such that the window is substantially equal to the pixel size of the sample (e.g., the pixel size of the sample plus a few pixels), and such that at the particular point in time the center of the window corresponds substantially to the location of the center of the sample. When the particular point in time occurs, step  1112  is performed. In step  1112 , the window receives emissions from the sample and then controller  110  windows image sensor  108  so that image data from the window is output to a data buffer. Preferably, in step  1112  image sensor  108  is windowed such that only the image data from the window is output to the data buffer. This image data comprises data from which the intensity of emissions from the sample can be determined. In step  1114 , the image data may be processed by image processing system  112 . 
     As illustrated in  FIG. 11 , steps  1111  and  1112  may be repeated. 
     Process  1100  is pictorially illustrated in  FIG. 12 , which shows a first window  1201  of pixel array  400  and a second window  1202  of pixel array  400 . Window  1201  is defined the first time step  1111  is performed and window  1202  is defined the second time step  1111  is performed. As shown in  FIG. 12 , a window follows the sample by keeping track of the location of the sample. Thus, by keeping track of the location of the sample, one need not read out the entire pixel array  400  in order to obtain information about a sample, rather one need only read out a small window of the pixel array. 
     While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments. 
     Additionally, while the processes described above are shown as a sequence of steps, this was done solely for the sake of illustration. Accordingly, it is contemplated that some steps may be added, some steps may be omitted, and the order of the steps may be re-arranged. 
     Additional features are disclosed in the document attached hereto as appendix A. 
     For the claims below the words “a” and “an” should be construed as “one or more.”