Abstract:
Apparatus and methods for size selecting nucleic acid molecules having wide range of applications including the production of DNA libraries for sequencing technologies. An automated high throughput system for size selection of multiple nucleic acid samples that uses imaging technique to detect the progress of a target fraction and feedback from the imaging to control electrophoresis. Predictive algorithms for timed nucleic acid extractions are generated to provide size selected nucleic acid molecules of required size ranges.

Description:
REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims Paris convention priority from U.S. application No. 61/497,586 filed on 16 Jun. 2011 and entitled Method and Apparatus for Automated Size Selection of Nucleic Acids which is hereby incorporated herein by reference for all purposes. For purposes of the United States of America, this application claims the benefit under 35 U.S.C. §119 of U.S. application No. 61/497,586 filed on 16 Jun. 2011 and entitled Method and Apparatus for Automated Size Selection of Nucleic Acids which is hereby incorporated herein by reference for all purposes. 
     
    
     TECHNICAL FIELD 
       [0002]    This invention relates to the automated size selection of nucleic acids. Aspects of the invention provide methods and apparatus useful for selecting nucleic acids according to size. 
       BACKGROUND 
       [0003]    There are a wide range of applications in which it is desirable to select nucleic acids, such as DNA or RNA by size. For example, size selection is used in the production of DNA libraries for use in sequencing and other applications. 
         [0004]    Various techniques for DNA size selection exist. Some of these techniques are undesirably labour intensive. One method for DNA size selection is to perform electrophoresis of a sample containing DNA in a gel. Since DNA of different sizes have different mobilities in the gel the electrophoresis separates the DNA into different bands by size. A band containing DNA in the desired size range can be identified and then manually cut out from the gel. The desired DNA can then be extracted from the gel. 
         [0005]    Some electrophoresis systems comprise wells formed in a gel. DNA can be run into the wells by electrophoresis. Invitrogen E-gels and the Lonza Flash Gel™ provide such wells. 
         [0006]    Y-channel size selection machines are another technology for DNA size selection. Examples are the Sage Pippin Prep™ and the Caliper XT™ machines. These machines can extract DNA of a desired size range from a sample by diverting DNA of the desired size range into a side channel and collecting the diverted DNA against a molecular weight cut-off filter. 
         [0007]    Solid Phase Reversible Immobilization Beads (SPRI) beads which are available from Beckman Coulter and others may be used to trap DNA of a certain size and then release the DNA after a wash and a change in pH. 
         [0008]    There remains a need for a DNA size-selection technology that can provide high throughput. There remains a need for a DNA size-selection technology that can provide accurate DNA size selection with reduced labour. 
       SUMMARY 
       [0009]    This invention has a number of aspects that may be applied together. Some of the aspects have independent application. One aspect provides apparatus for automated size-selection of nucleic acids. Another aspect provides a computer system for controlling apparatus for automated size-selection of nucleic acids. Another aspect provides methods for automated size-selection of nucleic acids. The nucleic acids may comprise DNA and/or RNA. Another aspect provides cartridges useful inter alia for automated size selection of nucleic acids. 
         [0010]    In one example embodiment, nucleic acids are size selected by loading DNA samples individually into agarose channels, each of which has a loading well at one end of the channel and an extraction well downstream. Electrophoresis is performed on the nucleic acids after loading and the nucleic acids are separated by size as they migrate towards the extraction well. The channel is imaged at regular intervals during this process and a software algorithm uses the images to identify reference bands and predict the time at which the desired nucleic acid fragments will arrive at the extraction well. The channel current is also individually controllable via pulse width modulation of the DC voltage so that if adjacent samples are running at different speeds, the extractions times can be altered so that no two samples need to be extracted at the same moment. 
         [0011]    Another aspect of the invention provides methods for size-selection of nucleic acids such as DNA, RNA and the like. Such methods comprise moving nucleic acids from a sample along a channel by electrophoresis; automatically monitoring progress of a reference fraction of the nucleic acids along the channel; based on the monitoring, estimating an estimated time of arrival of a target fraction of the nucleic acids at an extraction well in the channel; and extracting fluid containing the target fraction from the extraction well at the estimated time of arrival. The reference fraction may be the same as or different from the target fraction. For example, in some embodiments the reference fraction may comprise nucleic acids that are abundant in the sample (either originally present in the sample or added to the sample as a size marker) and the target fraction may comprise nucleic acids having sizes different from that of the reference fraction. Progress of the target fraction along the channel may be inferred from progress of the reference fraction. For example, the target fraction may be known to lead or lag behind the reference fraction by a certain percentage. In some embodiments, the target fraction of the nucleic acids comprises an adapter joined to a nucleic acid molecule of interest, and the reference fraction of the nucleic acids comprise the adapter which is not joined to the nucleic acid molecule of interest. In some embodiments, the methods may comprise automatically monitoring progress of a plurality of reference fractions of the nucleic acids along the channel. The plurality of reference fractions may comprise a DNA or RNA ladder of known sizes. 
         [0012]    In some embodiments the monitoring comprises, at spaced apart times, obtaining images of the channel and identifying areas in the images corresponding to the reference fraction. The images may, for example, be acquired by a camera mounted to view the channel. The camera may image a large number of channels simultaneously. Progress of the reference fractions (which are not necessarily the same for different channels) in multiple channels may be monitored using the same set of images. The estimated time of arrival of the target fraction may be estimated in some cases based on an average velocity of the target fraction based on differences between the positions of the reference fraction in two or more of the images. The images may comprise high dynamic range images. For example, the images may be obtained using a high dynamic range sensor or may be assembled from two or more different exposures. In some embodiments the images have a bit-depth of 10-bits or 12-bits or more. In some embodiments obtaining each of the images comprises operating an imaging device to obtain a plurality of different exposures of the channel and combining the plurality of different exposures to yield the image, wherein the image has a greater dynamic range than any of the plurality of different exposures. 
         [0013]    Some embodiments comprise specifying a size or size range of the target fraction. For example, the size or size range of the target fraction may be specified in absolute terms or relative to one or more of the reference fractions. For example, the size or size range of the target fraction may be specified as leading or lagging behind the reference fraction by a certain percentage. Some embodiments comprise scheduling a time of arrival for the target fraction at the extraction well; comparing the scheduled time of arrival to the estimated time of arrival and adjusting one or more electrophoresis parameters of an electrophoresis signal based on any difference between the scheduled time of arrival and the estimated time of arrival. In such embodiments, target fractions in different channels may be caused to arrive at extraction wells at different times (facilitating extraction of the target fractions using a single mechanism such as a robot carrying a pipetter that services each channel at the scheduled time). Also in such embodiments target fractions in different channels may be caused to arrive at extraction wells at the same time (facilitating extraction of the target fractions using a multi-channel mechanism such as a robot carrying a multi-channel pipetter that services several channels simultaneously at the scheduled time). 
         [0014]    Adjusting the one or more electrophoresis parameters may comprise adjusting a duty cycle of the electrophoresis signal, adjusting potentials of the electrophoresis signal or adjusting other parameters that define the electrophoresis signal. 
         [0015]    In some embodiments the method determines a location of an extraction well and/or a loading well in one or more channels by image analysis. This facilitates systems in which extraction wells in different channels are at different locations and also facilitates automatic compensation for variations in the positions of extraction wells and/or loading wells. 
         [0016]    Another aspect of the invention provides apparatus for size-selection of nucleic acids. The apparatus comprises: a channel having first and second ends and an extraction well in the channel; an electrophoresis power supply connected to deliver an electrophoresis signal to the channel to move nucleic acids from a sample along the channel; an imaging device mounted to image the channel; a controller connected to obtain images from the imaging device. the controller is configured to: automatically monitor progress of a reference fraction of the nucleic acids along the channel by analysis of the images; based on the monitoring, estimate an estimated time of arrival of a target fraction of the nucleic acids at the extraction well in the channel; and operate a mechanism to extract fluid containing the target fraction from the extraction well at the estimated time of arrival. 
         [0017]    The imaging device may comprise an electronic camera. The camera may be equipped with a filter that attenuates light outside of an emission band of a dye associated with the nucleic acid. 
         [0018]    In some embodiments the mechanism comprises a robotic system comprising a pipetter operable to transfer a sample into a loading well in the channel and to extract the fluid from the extraction well. In some embodiments the pipetter comprises a multi-channel pipetter capable of simultaneously introducing multiple samples into multiple channels or simultaneously extracting fluids from extraction wells in multiple channels. 
         [0019]    In some embodiments the channel comprises an elongated groove having opposed first and second sides and an electrophoresis medium in the groove and the first and second sides having steps extending longitudinally along the first and second sides, the electrophoresis medium filling the groove up to the steps. 
         [0020]    The electrophoresis medium may comprise, for example, a gel such as an agarose gel, an acrylamide gel, a denaturing acrylamide gel, or the like. 
         [0021]    In some embodiments the controller is configured to determine a location of the extraction well in the channel by image analysis of one or more of the images and to move the pipette tip to the determined location of the extraction well. 
         [0022]    In some embodiments the controller is configured to compare the estimated time of arrival of the target fraction at the extraction well to a desired time of arrival of the target fraction at the extraction well and to control the electrophoresis power supply to adjust one or more electrophoresis parameters of the electrophoresis signal based on any difference between the desired time of arrival and the estimated time of arrival. 
         [0023]    In some embodiments the controller is configured to control a rate of movement of the nucleic acids along the channel by proportional feedback control of the one or more electrophoresis parameters based on an error signal comprising a difference between the estimated time of arrival and a desired time of arrival of the target fraction at the extraction well. 
         [0024]    In some embodiments the controller comprises a scheduler configured to generate the desired time of arrival of the target fraction at the extraction well. 
         [0025]    The apparatus may comprise a proportional feedback controller configured to control the electrophoresis power supply to vary an average speed of the target fraction along the channel in response to an error signal representing a difference between the estimated time of arrival of the target fraction at the extraction well and a desired time of arrival of the target fraction at the extraction well. In some embodiments the controller is configured to reduce a difference between the estimated arrival time and the desired arrival time by temporarily interrupting application of the electrophoresis signal to the channel. 
         [0026]    Another aspect of the invention provides a cassette for use in size selection of nucleic acids. The cassette comprises a plate having a channel formed in the plate, the channel comprising an elongated groove having opposed first and second sides and an electrophoresis medium in the groove, first and second sides having steps, the electrophoresis medium filling the groove up to the steps. The plate may have one or more holes, grooves or other features for locking the plate into a known location relative to a robot. The channel may include both a loading well and an extraction well at spaced apart locations along the channel. The plate may optionally be transparent at least in its portion below the channel. 
         [0027]    Further aspects of the invention and features of example embodiments of the invention are illustrated in the accompanying drawings and/or described below. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0028]    The accompanying drawings depict non-limiting example embodiments of the invention. 
           [0029]      FIG. 1  illustrates a method for size-selecting a nucleic acid according to an example embodiment. 
           [0030]      FIG. 1A  illustrates an alternative example method. 
           [0031]      FIG. 2  illustrates schematically an image of one channel. 
           [0032]      FIG. 2A  is a plot illustrating density as a function of position along channel. 
           [0033]      FIG. 3  illustrates a method for identifying a peak corresponding to DNA of a predetermined size. 
           [0034]      FIG. 4  illustrates apparatus according to an example embodiment. 
           [0035]      FIG. 5  shows an example robot. 
           [0036]      FIG. 5A  shows an example deck. 
           [0037]      FIG. 5B  shows an example camera assembly. 
           [0038]      FIG. 6  is a screen shot of an example graphical display. 
           [0039]      FIG. 7  is a plan view of an example channel plate. 
           [0040]      FIG. 7A  is a cross section of an individual channel. 
           [0041]      FIG. 7B  shows an example comb useful for forming loading or extraction wells. 
           [0042]      FIG. 7C  is a plan view of an example channel plate according to another embodiment. 
           [0043]      FIG. 7D  is a perspective view of a channel plate with combs engaged for forming source and extraction wells. 
       
    
    
     DESCRIPTION 
       [0044]    Throughout the following description specific details are set forth in order to provide a more thorough understanding to persons skilled in the art. However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense. 
         [0045]    One aspect of the invention provides an automated method for size selection of multiple nucleic acid samples. The method uses imaging in conjunction with predictive algorithms to time extractions and provide size selected nucleic acids of a desired size range. The method can be practiced to advantage in conjunction with automated apparatus comprising one or more electrophoresis channels, a camera which acquires images of the one or more electrophoresis channels and a robot comprising a pipetter for introducing samples into corresponding channels and extracting size-selected nucleic acids from the channels. The channels may be filled, for example, with an agarose gel or an acrylamide gel. The channels may each have a loading well in the channel and an extraction well spaced apart from the loading well along the channel. 
         [0046]    The following description explains construction and operation of example embodiments being used to size-select DNA. For example, the DNA may comprise cDNA derived from RNA. The DNA to be size-selected may have a size in the range of 10 bp to 10 kbp. However, the invention may be applied to size-selection of other nucleic acids such as RNA. In some embodiments the nucleic acids comprise sheared nucleic acids. 
         [0047]      FIG. 1  illustrates a method  10  according to an example embodiment of the invention. In block  11  a sample containing DNA is introduced into a loading well in a channel containing a medium through which the DNA can be moved by electrophoresis. The sample containing the DNA may also comprise a dye (e.g., SYBR Green™ dye or ethidium bromide) which is introduced into the loading well together with the DNA. The function of the dye is to facilitate the detection or imaging of the DNA in the medium. In some embodiments, the DNA molecules in the sample may comprise an adapter, which may be useful for downstream applications such as DNA sequencing. The medium may, for example, comprise an agarose or acrylamide gel. In block  12  electrophoresis is commenced. Electrophoresis may be performed by applying an electrical potential difference between electrodes at opposing ends of the channel. The potential difference may comprise a DC electrical potential, a pulsed DC electrical potential or an unbalanced AC electrical potential, for example. The applied electrical potential drives the DNA to migrate from the loading well along the channel toward an extraction well. DNA of different sizes has different mobilities in the channel and so the DNA becomes size segregated. 
         [0048]    Optional block  13  provides a delay to allow the DNA to migrate far enough along the channel that concentrations of DNA of different sizes can be detected. Block  14  comprises determining the location along the channel of target DNA of a desired size range. In some embodiments, block  14  comprises obtaining a sequence of images of the channel with a camera, detecting one or more landmarks in the image(s) corresponding to DNA of one or more known sizes, and determining the position of the target DNA based on the position(s) of the landmark(s). The output of block  14  is a sequence of positions along the channel of the target DNA. 
         [0049]    Sizing reference(s) (e.g. landmarks) may be specified at or before run time; whether it is a DNA ladder, or an inherent feature expected to be present in the electropherogram. If the size reference used is a DNA ladder, the DNA ladder may be added to the sample prior to loading the sample into the loading wells. In some embodiments, the size reference is inherently present in the sample. For example, to size-select a cDNA sample derived from miRNA, the sample may comprise both cDNA+adapter fragments (e.g., having a size of 109 bp) as well as adapter-adapter fragments (e.g., having a size of 80 bp). The adapter-adapter fragments may be used as a size reference. 
         [0050]    The size-range of the target DNA may be specified in various ways, for example in absolute terms or relative to given reference(s), allowing for excision of fractions with sizes either dependent or independent of the electropherogram profile of the input sample. For example, if the input sample is sheared genomic DNA with an expected peak centre at ˜250 bp, the mobility range of the target fraction may be specified relative to the peak centre (e.g. 110%-90% of the peak-centre mobility), or the target may be specified as a absolute size range (e.g. 150 bp-200 bp) independent of the actual mobility of the peak centre. 
         [0051]    Block  15  determines an average velocity of the target DNA along the channel. Block  15  may, for example, be as simple as dividing a difference of two positions of the target DNA by an elapsed time between the images from which the positions were determined. Block  15  may take into account more than two positions. For example, block  15  may average or find the median of a plurality of velocities. 
         [0052]    Block  16  estimates a time of arrival of the target DNA at the extraction well. This determination may be based on a known, predetermined location of the extraction well. In some embodiments the location of the extraction well is determined in block  17  by locating the image of the extraction well in the images obtained in block  15  (or else separate images obtained for the purpose of locating the extraction well). This image recognition may be model-based (i.e. it is known in advance what the image of a channel is expected to look like in an image, where each channel is expected to be found in the image, and what the image of an extraction well is expected to look like in the image. Locating the extraction well may, for example, comprise, finding a location in the image where the correlation with a model image of an extraction well is maximized. In other simpler embodiments locating the extraction well in the image comprises locating one or more edges corresponding to the extraction well in the image. 
         [0053]    For example, the estimated time of arrival may be obtained by adding an estimated travel time to a current time. The estimated travel time may be determined, for example, by dividing a distance between the location of the extraction well and the current location of the target DNA by the current velocity of the target DNA as estimated in block  15 . 
         [0054]    Block  18  extracts the target DNA from the extraction well at the estimated arrival time. Block  18  may, for example comprise controlling a robot carrying a pipetter to place the pipetter into the extraction well at or before the estimated arrival time and withdrawing fluid from the extraction well into the pipetter at the estimated arrival time. The robot may then dispense the collected fluid into a reservoir where the fluid may be held or delivered for further processing. 
         [0055]    Method  10  has a range of variations. In some embodiments information regarding the velocity and/or position of the target DNA is applied to control the velocity of target DNA. Position information obtained from run-time electropherograms and time information is used in a feedback loop to control electrophoresis speed of the target DNA fraction in addition to its arrival time at the extraction well. 
         [0056]    Feedback control may be applied, for example, to adjust the estimated arrival time of the target DNA at the extraction well. The estimated arrival time may be adjusted, for example, adjusting a duty cycle and/or voltage of an electrophoresis field and/or by pausing application of the electrophoresis field one or more times. Embodiments which adjust velocity of the target DNA by adjusting duty cycle can be advantageous since the relationship between duty cycle and velocity tends to be linear or nearly linear. This simplifies control. Electrophoresis speed and extraction scheduling may be simultaneously controlled for an arbitrary number of samples. In one embodiment, extraction scheduling processes 96 samples running in parallel. 
         [0057]      FIG. 1A  illustrates an alternative example method  10 A which is similar to method  10  except that it includes a block  19  that schedules a scheduled arrival time for the target DNA and a block  20  that compares the estimated arrival time from block  16  to the scheduled arrival time for the target DNA. Block  21  receives a control signal from block  20  and adjusts electrophoresis parameters based on the control signal. Loop  22  may be repeated periodically at a rate sufficient to control the progress of the target DNA so hat the target DNA arrives at the extraction well at the scheduled time. 
         [0058]    In block  21 , the electrophoresis parameters may be adjusted, as appropriate, to retard the progress of the target DNA, to accelerate the progress of the target DNA or to maintain the current rate of progress of the target DNA. In some embodiments the adjustment depends merely on the sign of the control signal (i.e. whether the estimated arrival time is before or after the scheduled arrival time). In other embodiments the adjustment is based at least in part on a magnitude of the difference between the estimated arrival time and the scheduled arrival time (or equivalently a magnitude of the difference between an estimated velocity and a velocity that would result in the target DNA arriving at the extraction well at the scheduled time. 
         [0059]    In typical cases the target DNA does not have a specific size but has a range of sizes. Thus the target DNA will arrive at an extraction well during a time window having a length determined by the range of sizes in the DNA fraction as well as by the electrophoresis parameters. In some embodiments a target fraction may be specified initially and also continuously adjusted until the fraction is extracted. 
         [0060]    Control over the time at which target DNA arrives at an extraction well may be applied to good effect in the case where multiple electrophoresis channels are being operated at the same time. For example, electrophoresis of DNA in each of a plurality of channels may be controlled to cause target DNA in each channel to arrive at an extraction well at a scheduled time such that the scheduled times in different channels are different. The target DNA in different channels may be the same or different. This can facilitate using a robot to extract target DNA from each of the channels without requiring the same pipetter of the robot to be extracting fluid from two extraction wells at the same time. The scheduled times may be assigned to ensure that there is enough time for the robot to make each of the scheduled extractions. 
         [0061]    Control over the electrophoresis velocity may be applied to compensate for variations between channels in electrophoresis velocity (caused, for example, by inhomogeneities in the electrophoresis medium or other differences in the electrophoresis medium used in different channels. The control may also be applied to compensate for differences in the location of the extraction well between channels. The control may also be applied to compensate for differences in the target DNA for different channels. 
         [0062]    Some embodiments employ a multi-channel robot. For example, such a robot may have a plurality of pipetters arranged so that their tips can be simultaneously inserted into a plurality of extraction wells. For example, the robot may carry 8, 16 or some other number of pipetters. In some such embodiments channels are arranged side-by-side and the robot may be configured to simultaneously introduce fluid into N adjacent loading wells or to simultaneously remove fluid from N adjacent extraction wells. 
         [0063]    In some embodiments which use a multi-channel robot, electrophoresis in a plurality of channels is controlled to cause target DNA in the plurality of channels to reach extraction wells at the same scheduled time. The target DNA may differ among the plurality of channels. The robot may then be controlled to place pipette tips into the extraction wells of the plurality of channels at the scheduled time and to simultaneously extract fluid from the extraction wells. Such embodiments permit different scheduled arrival times to be assigned to groups of channels. Electrophoresis in the individual channels in each group may be separately controlled to cause target DNA in each channel in the group to arrive at the corresponding extraction well at the time scheduled for that group. The scheduled times for different groups of channels may be spaced apart such that the robot has time to make the scheduled extractions. Such embodiments can provide high throughput electrophoresis. 
         [0064]    The principles described above may also be applied in situations where it is desired to extract two or more fractions from the same sample. For such applications, electrophoresis may be performed to bring a first target fraction to an extraction well and to extract the first target fraction. Subsequently, further electrophoresis may be performed to bring a second fraction to the extraction well. The second fraction may then be extracted. If desired, the first and second target fractions may be kept isolated from one another. For example, each of the first and second target fractions may be transferred from the extraction well to a separate destination well. It is also possible to transfer multiple fractions from the same sample to the same destination well if that is desired. 
         [0065]    In some applications, three or more fractions may be extracted from the same sample. Where two or more target fractions are to be extracted from the same sample then extraction of each of the target fractions may be separately scheduled. After a first target fraction has been extracted from a channel at a first scheduled time, electrophoresis parameters for the channel may be controlled to bring the second target fraction to the extraction well for extraction at a second scheduled time. 
         [0066]    In some embodiments electrophoresis is controlled in a plurality of channels to bring a corresponding plurality of first target fractions to the extraction wells in the plurality of channels at a first time. A multi-channel pipetter or other multi-channel extraction mechanism may then be applied to transfer the first target fractions to corresponding destination wells. Electrophoresis in the plurality of channels may then be controlled to bring second target fractions to the extraction wells in the channels at the same time. It is not necessary that the spacing between the first target fractions and the second target fractions be the same between the different channels. Electrophoresis may be controlled to move the second target fraction toward the extraction well faster in some channels than in others. In some embodiments electrophoresis is controlled to bring the second target fractions to extraction wells at the same time in the plurality of channels so that the second target fractions can be simultaneously extracted using the multi-channel pipetter. 
         [0067]      FIG. 2  illustrates schematically an image of one channel  24 . Channel  24  comprises a strip  25  of a suitable electrophoresis medium with a buffer reservoir  25 A,  25 B at each end. A loading well  26 A is located in medium  25  near buffer reservoir  25 A. An extraction well is located in medium  25  at a location that is spaced apart from loading well  26 A toward buffer reservoir  25 B. 
         [0068]    Also shown in  FIG. 2  are various bands of DNA that have been carried along medium  25  from loading well  26 A by electrophoresis. Because DNA of different sizes moves at different rates under electrophoresis, the bands at different locations represent DNA of different sizes. Different bands may have different densities in the image. The bands may all represent DNA that is present in a sample. In some embodiments DNA of a known size or a set of known sizes (e.g. a DNA ladder) may be added to the sample for the purpose of providing a size scale that may be used to determine the location of target DNA. 
         [0069]    In some embodiments sizing references such as DNA ladders are run in the same channel  24  as input samples. This ensures sizing accuracy in comparison to embodiments where sizing references and samples are run in separate channels. 
         [0070]      FIG. 2A  is a plot illustrating density as a function of position along channel  24 . Peaks in curve  27  correspond to the locations of the bands shown in  FIG. 2 . The methods described above may identify a peak in curve  27  corresponding to the target DNA or infer a current position of the target DNA from locations of one or more other peaks corresponding to DNA having a known size relationship(s) to the target DNA. 
         [0071]    Some embodiments provide a scheduler. The scheduler may, for example, be implemented in software. The scheduler may schedule: the transfer of samples into source wells  26 A in channels  24 , commencement of electrophoresis in channels  24  and the extraction of target fractions from extraction wells  26 B. In some embodiments the scheduler operates while samples are being run in channels  24  and may re-schedule extraction of target fractions in response to the monitoring of the progress of the target fraction (or a band having a known relationship to the target fraction). The schedule may initially schedule extraction of a target fraction in a time-slot that is separated from a time of commencement of electrophoresis in a channel by a period that is longer than the shortest period in which a target fraction could possibly progress from the source well  26 A to the corresponding extraction well  26 B. The time period used for this initial scheduling may be determined based on a measured distance from the source well  26 A to the extraction well  26 B in some embodiments. The time period may be generated based on an assumed average velocity of the target fraction that is less than a maximum velocity achievable within an available range of electrophoresis parameters. The assumed average velocity may be a function of a size of the target fraction and the characteristics of the medium in which electrophoresis is being performed. 
         [0072]    In some embodiments, the length of a period scheduled by the scheduler for extraction of a target fraction is variable and depends on the sizes of nucleic acid included in the target fraction (a target fraction which includes a greater range of sizes will take longer to extract than a target fraction in which the spread of sizes is small). In some embodiments start and stop times for extraction of a target fraction are adjusted on the basis of the estimated times of arrival at the extraction well of leading and trailing edges of the target fraction. 
         [0073]    In some embodiments the scheduler monitors for conflicts between times for extraction of target fractions from different channels  24 . In some such embodiments, in the case of a conflict (i.e. periods assigned to extraction of target fractions from different channels  24  overlap the scheduler may revise the scheduled time for extraction of the target fraction from one of the channels  24  to remove the conflict. Changing of the scheduled extraction time may automatically result in parameters of the electrophoresis in the rescheduled channel  24  being altered so as to control the progress of the target fraction in the channel  24  so that the target fraction arrives at the extraction well at the rescheduled time. 
         [0074]    In some embodiments the electrophoresis parameters for a channel  24  are controlled such that upon the leading edge of the target fraction arriving at the corresponding extraction well  26 B and extraction commencing, the rate of electrophoresis is increased, thereby reducing the time over which extraction must be continued to extract the entire target fraction. 
         [0075]      FIG. 3  illustrates a method  30  for identifying a peak corresponding to DNA of a predetermined size in channel  24 . Block  32  applies electrophoresis to a channel using known electrophoresis parameters for a period of time. Block  33  obtains an image of the channel at the end of block  33 . Block  34  identifies a range of positions along the channel based upon known characteristics for the target DNA. For example, a predetermined calibration curve  35  may be provided which relates position along the channel to DNA size. In some embodiments a plurality of different calibration curves  35  are provided. The different calibration curves may apply to different media that may be used in channels  24 . 
         [0076]    Block  34  estimates a position or range of positions in which target DNA is expected to be found. The estimated position may be a function of the length of time that electrophoresis has been performed, the medium in channel  24 , the electrophoresis parameters and the characteristics (especially size) of the target DNA. An operator may enter a size or size range for the target DNA. Block  34  may use an appropriate calibration curve  35  to identify the expected position of a peak in curve  27  corresponding to the target DNA. Block  36  sets a range  37  (see  FIG. 2A ) and searches curve  27  for a peak within range  37 . If a peak is successfully detected (as determined e.g. by a YES result from block  39  then the peak is identified as the initial location of the target DNA. Once a peak corresponding to target DNA has been identified in one image the peak may be tracked through subsequent images as it propagates along the channel. Prominent features of the electropherogram profile may be identified at run-time and used to help maintain size integrity as faster-moving size references move out of the field of view. 
         [0077]    Method  30  may be applied to identify one or more peaks corresponding to DNA in a DNA ladder and/or a sample. In some embodiments one or more peaks that are different from the target DNA are identified and tracked as described above. A current location of the target DNA may be identified relative to such peaks. For example, a user may specify the amount by which target DNA is expected to lead or lag one or more such peaks. 
         [0078]      FIG. 4  illustrates apparatus  40  according to an example embodiment. Apparatus  40  comprises a robot  42  comprising a pipetter  44  that can be positioned by robot  42  over desired locations in a field  43 . For example, robot  42  may comprise an XYZ stage that supports a single-channel pipette pump, which also supports a buffer loading line and tip ejection mechanism. A source plate  45  comprises a plurality of source wells  45 A. A destination plate  46  comprises a plurality of destination wells  46 A. A plurality of channels  47  is provided within field  43 . 
         [0079]    Robot  42  comprises a controller  42 A that can control the position of pipetter  44 . Controller  42 A may, for example, control robot  42  to load a channel by: picking up a pipette tip  48  at a station  48 A, positioning the pipette tip over a selected source well  45 A, lowering the pipette tip into the source well  45 A and drawing fluid into the pipette tip  45 A, raising the pipette tip and repositioning it over a loading well of a selected channel  47 , lowering the pipette tip into the loading well, operating the pipetter to dispense the fluid into the source well, raising the pipette tip and moving to pipette tip to a storage area for used pipette tips and disconnecting the used pipette tip. 
         [0080]    Controller  42 A may, for example, control robot  42  to retrieve target DNA from a channel by: picking up a pipette tip  48  at station  48 A, positioning the pipette tip over the extraction well in the selected channel, just prior to the estimated arrival of the target DNA lowering the pipette tip into the extraction well of the channel, drawing fluid into the pipette tip  45 A over a period of time corresponding to the expected arrival of the target DNA, raising the pipette tip and repositioning it over a destination well  46 A, lowering the pipette tip into the destination well, operating the pipetter to dispense the fluid into the destination well, raising the pipette tip and moving to pipette tip to a storage area for used pipette tips and disconnecting the used pipette tip. 
         [0081]    Apparatus as described herein may be configured to process any sensible number of samples. In some embodiments, apparatus as described herein provides automated size selection for 96 samples concurrently. In other example embodiments apparatus process multiples of 96 samples concurrently. Other example embodiments are configured to process other numbers of samples. 
         [0082]    Robots suitable for use as robot  42  are commercially available. Robots suitable for use as robot  42  may also be made from commercially-available components in ways known to those of skill in the art. 
         [0083]    Apparatus  40  comprises an imaging device  50  which may, for example, comprise a camera arranged to obtain images of channels  47 . Imaging device  50  may comprise a high dynamic range imaging device. For example, camera  50  or a controller connected to receive images from camera  50  may be configured to obtain and combine images taken at different exposure times to expand the detectable dynamic range. This allows dim bands to be visible without saturating the brightest bands. 
         [0084]    A light source  52  illuminates channels  47  to facilitate imaging of nucleic acids propagating along the channels. Where the DNA is associated with a dye the light source may emit light corresponding to an absorption band of the dye (e.g. a band corresponding to a wavelength that excites a fluorophore of the dye. Light source  52  may comprise a filter that blocks wavelengths outside of this range. For example, SYBR Green™ dye absorbs light at 488 nm. The light source may emit blue light. For example, the light source may comprise an array of blue LEDs. Alternatively or additionally, the light source may emit UV light. Camera  50  may include a filter that preferentially admits fluorescence of the dye. for example, SYBR Green™ dye emits light at 520 nm. The camera may have a bandpass or notch filter that passes light at 520 nm but attenuates light at other wavelengths. 
         [0085]    For example, the camera may be fixed to a component of robot  42  such that the camera is at a fixed distance from channels  47 . In a prototype embodiment camera  50  and LED illuminator  52  are fixed to a Y-axis arm of robot  42 . 
         [0086]    A multi-channel electrophoresis power supply  54  is configured to provide electrophoresis potentials across channels  47 . Power supply  54  may comprise a single unit or a plurality of separate units. A controller  55  is connected to receive images from camera  50  to control power supply  54  and to coordinate actions of robot  42 . A user interface  56  allows users to provide control inputs and information to guide operation of apparatus  40 . 
         [0087]    In operation of an example system, source and destination plates  45 ,  46  are loaded along with two tip boxes containing pipette tips. Plates comprising channels  47  are set on the deck and electrode arrays are placed so that their electrodes are in electrical contact with channels  47  in the ends of the channel plate. In some embodiments the electrodes are mounted to a structure which permits them to be introduced into buffer wells at each end of each channel. For example, the apparatus may comprise a hinged frame carrying first and second electrodes corresponding to each channel. The first and second electrodes may be mounted on the hinged frame and the hinged frame may be movable between a first position wherein the first and second electrodes project into first and second buffer reservoirs of a channel and a second position wherein the first and second electrodes are removed from the first and second buffer reservoirs. 
         [0088]    The control software is configured with the location of the samples (a whole 96 well plate or less or more), type of samples and positions of the channel plate(s). 
         [0089]    When a run commences, buffer wells in the channel plates are filled. The samples are loaded sequentially (e.g. by the robot into the loading wells in channels  47 ) and electrophoresis commences. In one embodiment, the on board camera  50  is used to locate the extraction well in each channel  47  avoiding the requirement to manually configure the location of the extraction wells. This facilitates the possibility of providing extraction wells at different locations within their channels  47 . 
         [0090]    Some embodiments comprise a mechanism for measuring and/or setting the Y position of the pipette tip. Knowing the exact position of the pipette tip facilitates precise loading and retrieval of nucleic acid samples in small wells. Such a mechanism is useful because the ends of different pipette tips can be at somewhat different locations relative to the robot when mounted to the pipetter. In an example embodiment the mechanism comprises a switch (which can be for example a microswitch, proximity switch or the like) that changes state when a pipette tip is in a predetermined location relative to the switch. The switch may be at a convenient location in the field of the robot. 
         [0091]    In some embodiments, the switch is located near a supply of fresh pipette tips such that the Y position of each new pipette tip may be set by moving the robot to bring the pipette tip against the switch. In such embodiments the pipette tip may be positioned near to the switch and then moved toward the switch in the Y direction until the switch changes state. This mechanism may be used to individually measure the location of the end of each pipette tip after a tip is loaded. The measured location may be used to compensate for slight misalignments in different pipette tips. The illustrated system  40  comprises a switch  49  arranged to switch when a pipette tip presses against the switch in a Y direction (a direction parallel to channels  47 ). 
         [0092]      FIG. 5  shows an example robot.  FIG. 5  shows a lower deck which accommodates controllers and power supplies, and an upper deck which accommodates channel plates and electrodes. Above that is the pipetting head with pump, buffer delivery system and camera and lights for imaging the channels. 
         [0093]      FIG. 5A  shows an example deck.  FIG. 5A  shows deck locator plates that hold the deck in position. Tip boxes, source places, destination plates and channel plates are all mounted to the deck. At least the source places, destination plates and channel plates are removable from the deck. Spring pins are provided to hold the plates against locator pins so that source wells, destination wells and channels will be in known positions when the plates are installed on the deck. 
         [0094]      FIG. 5B  shows an example camera system comprising a camera  50  and LED arrays  52 . LED arrays  52  comprise blue light emitting light sources such as blue LEDs of LEDs covered by blue filters in some embodiments. 
         [0095]    In an example embodiment, controller  55  comprises a processor configured to execute instructions provided in software. The software creates a run protocol (which sample runs in which channels, in which order, and what destination wells the respective extractions will end up in) based on data input by the user. This is communicated to the user graphically. 
         [0096]      FIG. 6  is a screen shot of an example graphical display.  FIG. 6  shows the display mid-run. Samples have been loaded sequentially starting in the lower left and the first two and half plates have completed runs. The remaining samples to the right are running and each channel&#39;s status is shown graphically based on the most recent image. The plot at top is an electropherogram for a channel selected by the user, showing a size reference peak  59 A and target region  59 B. 
         [0097]    Another aspect of the invention that may be used together with a robot as described above but also may have other applications provides channel plates for use in separation of nucleic acids. In some embodiments one or more channels is provided on a plate. The plate may be removably placed within the field of a robot as described above, for example. Providing DNA separation media in channels as opposed to slabs (e.g. slab gels) has the advantage that the possibility of cross-contamination from one sample to another is reduced. 
         [0098]      FIG. 7  is a plan view of an example channel plate  80 . Plate  80  comprises location features  81  such as holes (see  FIG. 7A ) or notches (see  FIG. 7C ) for receiving locating pins or other locating features that permit plate  80  to be repeatably positioned in the field of a robot or other apparatus. A plurality of channels  24  extend along plate  80 . Each channel  24  comprises a strip  25  of a suitable electrophoresis medium. A buffer reservoir  25 A,  25 B is provided at each end of strip  25 . A loading well  26 A is located in strip  25  near buffer reservoir  25 A. An extraction well  26 B is located in strip  25  at a location that is spaced apart from loading well  26 A toward buffer reservoir  25 B. In some applications extraction wells  26 B of different channels are aligned with one another but this is not mandatory. In some applications it may be convenient to provide extraction wells  26 B that are at different locations along strip  25  in different channels  24 . 
         [0099]      FIG. 7A  is a cross section of an individual channel  24 . Channel  24  optionally has a small step edge on either side of strip  25 . In the illustrated embodiment steps  83  are shown. Steps  83  provide corners  84 . Corners  84  run length-wise along strip  25  parallel to one another. In the illustrated embodiment, corners  84  are parallel to flat top and bottom surfaces  84 A and  84 B of plate  80 . Medium  86  (for example an agarose gel, an acrylamide gel or the like) fills strip  25  up to the level of corners  84 . Steps  83  help to make the top surface of material  86  in strip  85  flat along the length of strip  25 . The presence of corners  84  as material  86  is introduced into strip  25  helps to reduce the tendency of surface tension of material  86  to form a meniscus at the surface of material  86 . Optional features such as small divots or dimples  89  (See  FIG. 7C ) may be formed in walls of strip  25  near the ends of strip  25  in order to mechanically lock material  86  in place. 
         [0100]    Dimensions of channel  24  may be varied. In an example embodiment, strip  25  has a depth in the range of about 6 to 12 mm, preferably 8 to 10 mm. In an example embodiment, strip  25  has a width of 3 mm to 11 mm, preferably 4 mm to 7 mm. The principles described herein may be applied, however, to channels of other dimensions. 
         [0101]    Plate  80  may be made of a suitable plastic or other electrically-insulating material. In some embodiments plate  80  is injection molded however, plate  80  may also be fabricated by machining or in any other suitable manner. 
         [0102]    A plate  80  may be prepared by temporarily damming or filling buffer reservoirs  25 A and  25 B and pouring a suitable amount of a settable material  86  into strips  25 . Preferably the entire volume of each buffer reservoir is filled while material  86  is cast so that material  86  is unable to flow into the buffer reservoirs. For example, an agarose gel may be poured into strips  25  while the gel is in a liquid form and then allowed to set in strips  25 . The amount of material  86  introduced into each strip may be just enough that a surface of the material is at the level of corners  84 . 
         [0103]    Loading and extraction wells may be formed in material  86  while the material is being cast into strips  25 . In other embodiments the loading and/or extraction wells may be formed after material  86  has set. In some embodiments loading and/or extraction wells are formed by placing loading and/or extraction combs at appropriate locations along strips  25 .  FIG. 7B  shows an example comb  87 . Each comb  87  comprises a row of pins  87 A. A comb  87  may be placed on plate  80  transversely to strips  25  such that pins  87 A are arranged to project into strips  25  of the channels  24  crossed by the comb  87 . 
         [0104]    Plate  80  may comprise locating features  88  to place combs  87  in desired alignment for forming loading wells and/or extraction wells. Multiple sets of locating features  88  may be provided to facilitate forming extraction wells at different locations along strips  25 . As noted above, it can be desirable to provide extraction wells at a location that is tailored to the separation to be performed. The best length of separation channel between loading well  26 A and extraction well  26 B depends on the length of DNA or other target nucleic acid and the desired degree of separation. 
         [0105]    A comb  87  for forming extraction wells may have pins  87 A that are somewhat wider than the pins  87 A used to form loading wells. Providing loading wells  26 A that do not extend the full width of strips  25  helps to avoid loss of sample at the sides of a loading well. Extraction wells  26 B may extend the full width of strips  25  or nearly the full width of strips  25 . 
         [0106]    A range of embodiments provide channels in which loading wells are wider than extraction wells. In one particular example embodiment, the loading well has a dimension of 1.2×3.5×9 mm, and the extraction well has a dimension of 1.2×5.5×9 mm (i.e., 2 mm wider than the loading well). A loading well having a dimension of 1.2×3.5×9 mm allows a sample having a volume of up to 37.8 μl to be loaded. An extraction well having a dimension of 1.2×5.5×9 mm allows a volume of up to 59.4 μl to be withdrawn. 
         [0107]    Combs  87  may be designed so that pins  87 A that form the wells can ‘float’ slightly (e.g. about 0.25 mm) in their mounting frames. This facilitates removing combs  87  after material  86  has set. 
         [0108]    A plate  80  comprising one or more channels  24  may be provided in the form of a pre-prepared cassette provided in sterile packaging. The packaging may, for example, comprise a sterile cover that can be peeled off to reveal channels  24 . In some embodiments the cassette may be supplied with combs inserted into the loading and/or extraction wells. A user may remove the combs prior to use. 
         [0109]      FIG. 7C  is a plan view of an example channel plate according to another embodiment. 
         [0110]      FIG. 7D  is a perspective view of a channel plate with combs  87 - 1  and  87 - 2  inserted in preparation for casting an electrophoresis medium into channels  24 . Comb  87 - 1  may have narrower pins than comb  87 - 2  in some embodiments. 
         [0111]    although a camera provides a convenient tool for imaging a plurality of channels and simultaneously tracking progress of one or more reference fractions in each of the channels, other tools may be used in place of a camera. For example, a 1-D line scanner could be provided to measure a concentration of a nucleic acid as a function of position along a channel. Further, it is not mandatory that the camera view the channels from above. In some embodiments trays carrying the channels are transparent, at least in their parts underlying the channels and the camera views the channels from below through the plates. 
       Interpretation of Terms 
       [0112]    Unless the context clearly requires otherwise, throughout the description and the claims:
       “comprise,” “comprising,” and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.   “connected,” “coupled,” or any variant thereof, means any connection or coupling, either direct or indirect, between two or more elements; the coupling or connection between the elements can be physical, logical, or a combination thereof.   “herein,” “above,” “below,” and words of similar import, when used to describe this specification shall refer to this specification as a whole and not to any particular portions of this specification.   “or,” in reference to a list of two or more items, covers all of the following interpretations of the word: any of the items in the list, all of the items in the list, and any combination of the items in the list.   the singular forms “a”, “an” and “the” also include the meaning of any appropriate plural forms.       
 
         [0118]    Words that indicate directions such as “vertical”, “transverse”, “horizontal”, “upward”, “downward”, “forward”, “backward”, “inward”, “outward”, “vertical”, “transverse”, “left”, “right” , “front”, “back” , “top”, “bottom”, “below”, “above”, “under”, and the like, used in this description and any accompanying claims (where present) depend on the specific orientation of the apparatus described and illustrated. The subject matter described herein may assume various alternative orientations. Accordingly, these directional terms are not strictly defined and should not be interpreted narrowly. 
         [0119]    Embodiments of the invention may be implemented using specifically designed hardware, configurable hardware, programmable data processors configured by the provision of software (which may optionally comprise ‘firmware’) capable of executing on the data processors, special purpose computers or data processors that are specifically programmed, configured, or constructed to perform one or more steps in a method as explained in detail herein and/or combinations of two or more of these. Examples of specifically designed hardware are: logic circuits, application-specific integrated circuits (“ASICs”), large scale integrated circuits (“LSIs”), very large scale integrated circuits (“VLSIs”) and the like. Examples of configurable hardware are: one or more programmable logic devices such as programmable array logic (“PALs”), programmable logic arrays (“PLAs”) and field programmable gate arrays (“FPGAs”)). Examples of programmable data processors are: microprocessors, digital signal processors (“DSPs”), embedded processors, graphics processors, math co-processors, general purpose computers, server computers, cloud computers, mainframe computers, computer workstations, and the like. For example, one or more data processors in a control circuit for a device may implement methods as described herein by executing software instructions in a program memory accessible to the processors. 
         [0120]    Processing may be centralized or distributed. Where processing is distributed, information including software and/or data may be kept centrally or distributed. Such information may be exchanged between different functional units by way of a communications network, such as a Local Area Network (LAN), Wide Area Network (WAN), or the Internet, wired or wireless data links, electromagnetic signals, or other data communication channel. 
         [0121]    For example, while processes or blocks are presented in a given order, alternative examples may perform routines having steps, or employ systems having blocks, in a different order, and some processes or blocks may be deleted, moved, added, subdivided, combined, and/or modified to provide alternative or subcombinations. Each of these processes or blocks may be implemented in a variety of different ways. Also, while processes or blocks are at times shown as being performed in series, these processes or blocks may instead be performed in parallel, or may be performed at different times. In addition, while elements are at times shown as being performed sequentially, they may instead be performed simultaneously or in different sequences. 
         [0122]    Software and other modules may reside on servers, workstations, personal computers, embedded processors, process controllers, tablet computers, and other devices suitable for the purposes described herein. 
         [0123]    The invention may also be provided in the form of a program product. The program product may comprise any non-transitory medium which carries a set of computer-readable instructions which, when executed by a data processor, cause the data processor to execute a method of the invention. For example, the computer readable instructions may program a computer to control a robotic nucleic acid sizing system as described herein and/or to schedule operations in a nucleic acid sizing system as described herein. Program products according to the invention may be in any of a wide variety of forms. The program product may comprise, for example, non-transitory media such as magnetic data storage media including floppy diskettes, hard disk drives, optical data storage media including CD ROMs, DVDs, electronic data storage media including ROMs, flash RAM, EPROMs, hardwired or preprogrammed chips (e.g., EEPROM semiconductor chips), nanotechnology memory, or the like. The computer-readable signals on the program product may optionally be compressed or encrypted. 
         [0124]    In some embodiments, the invention may be implemented in software. For greater clarity, “software” includes any instructions executed on a processor, and may include (but is not limited to) firmware, resident software, microcode, and the like. Both processing hardware and software may be centralized or distributed (or a combination thereof), in whole or in part, as known to those skilled in the art. For example, software and other modules may be accessible via local memory, via a network, via a browser or other application in a distributed computing context, or via other means suitable for the purposes described above. 
         [0125]    Where a component (e.g. a software module, processor, assembly, device, circuit, etc.) is referred to above, unless otherwise indicated, reference to that component (including a reference to a “means”) should be interpreted as including as equivalents of that component any component which performs the function of the described component (i.e., that is functionally equivalent), including components which are not structurally equivalent to the disclosed structure which performs the function in the illustrated exemplary embodiments of the invention. 
         [0126]    Specific examples of systems, methods and apparatus have been described herein for purposes of illustration. These are only examples. The technology provided herein can be applied to systems other than the example systems described above. Many alterations, modifications, additions, omissions and permutations are possible within the practice of this invention. This invention includes variations on described embodiments that would be apparent to the skilled addressee, including variations obtained by: replacing features, elements and/or acts with equivalent features, elements and/or acts; mixing and matching of features, elements and/or acts from different embodiments; combining features, elements and/or acts from embodiments as described herein with features, elements and/or acts of other technology; and/or omitting combining features, elements and/or acts from described embodiments. 
         [0127]    It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions, omissions and sub-combinations as may reasonably be inferred. The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole. 
         [0128]    While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations.