Abstract:
The present invention describes sample loading devices for use in polyacrylamide gel electrophoresis systems. The sample loading devices comprise alternating areas of absorbent membranes and diffusion barriers, where the diffusion barriers are formed by the application of some form of energy, such as heat, pressure, laser energy, RF energy or the like. The present invention also describes devices and methods for making sample loading devices, and methods of loading samples into polyacrylamide gel electrophoresis systems.

Description:
RELATED APPLICATION 
     This application claims benefit to Provisional Application No. 60/113,801, filed Dec. 23, 1998, the entire teachings of which are incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     Polyacrylamide gel electrophoresis is one of the most powerful tools used in the field of biotechnology. By passing an electric current through a polyacrylamide gel, the polyacrylamide gel electrophoresis method separates samples, such as nucleic acids, proteins and other biologically relevant molecules, by charge, size, conformation or other characteristics of the sample. One of the critical procedures in performing polyacrylamide gel electrophoresis is gel loading, which involves the addition of samples to the gel. To facilitate the sample loading process, many techniques and devices have been used. However, the loading process still remains one of the most time consuming and technique dependent steps in the polyacrylamide gel electrophoresis process. 
     One techniques involves using multiple syringes or pipettes to load the sample directly on the gel. Another technique is to use a sample loading device that has a substrate and an absorbent membrane on which a plurality of samples are loaded. A method of “diffusion isolation” is needed to prevent the individual samples from intermixing. 
     In the use of the substrate/absorbent membrane technique, it is known to use a sample loading device referred to as a “comb” that has a plurality of fingers that can help maintain the integrity of individual samples. For ensuring “diffusion isolation” of each sample within the loading area of the comb, the width of the absorbent material corresponding to the gel loading area is physically separated by cutting the comb into numerous “fingers.” The length of the comb is constrained by the size of the gel and the corresponding “read” area of the apparatus which receives the comb, therefore the number of fingers and the physical separation between the samples is limited by this length of the comb. Furthermore, as the number of fingers and separating cuts is increased, the comb becomes less stable, making it more difficult to handle and place it into the gel. 
     Another embodiment of the sample loading device has scoring of the absorbent membrane (i.e., removing membrane material from the substrate) to produce barriers between the samples to maintain diffusion isolation. Another technique is creating diffusion isolation by use of hydrophobic ink on the absorbent material to produce barriers between the samples. The use of scoring or hydrophobic ink or a combination thereof is most effective when samples are resuspended in a water soluble solution; however, many loading buffers used for electrophoresis contain organic solvents that are unlikely to be impeded by a scored trough or by hydrophobic ink. 
     SUMMARY OF THE INVENTION 
     In the present invention, the properties of the absorbent membrane in a sample loading device are physically altered by pressure, heat, a combination of heat and pressure, or other such treatments, so that the regions between sample loading areas are unable to absorb the sample. The present method produces sample diffusion barriers that prevent sample diffusion and contamination with adjacent samples. The physical stability of the sample loading device is maintained because the membrane material remains continuous and lacks the unsupported “fingers” described in the prior art. Therefore, the present invention is more robust, making it easier to insert the sample loading device into a gel, thereby increasing data quality and resolution and reducing fabrication costs. Additionally due to the inherent and resultant properties of the absorbent and backing materials as a laminate, it is easier and more reliable to fabricate and apply standard quality and redundancy controls to the process/device than with prior art devices. 
     In one embodiment, the present invention describes a system that allows effective loading and scanning high resolution of about 48 or more samples in a standard polyacrylamide electrophoresis gel. The system produces results that are unexpectedly superior over prior art materials and methods. The sample loading device of the present invention also allows more samples to be loaded in a given area than other sample loading devices. 
     The sample diffusion barrier(s) of the present invention is formed utilizing the inherent properties of the absorbent membrane. The membrane is altered physically using pressure, conductive heat or convection heat (such as laser energy or RF energy), a combination of heat and pressure, or similar techniques. The material properties of the absorbent membrane are physically altered so that the absorbent membrane is changed into an effective, continuous diffusion barrier that is unable to absorb the sample. Thus, the sample loading device of the present invention with the homogenous altered absorbent membrane as the physical barrier between the sample loading areas, eliminates the need to add extraneous matter, such as hydrophobic inks, or to remove or prevent deposition of an absorbent material. 
     The present invention describes at least one sample loading device that comprises alternating areas of absorbent membranes and diffusion barriers. The diffusion barriers are formed by physically altering the absorbent membrane using heat, pressure, or combination pressure and heat, or other similar techniques. 
     At least one method of loading samples into a gel of an electrophoresis gel system is also described. The method includes applying a sample to a sample loading device, where the sample loading device comprises alternating absorbent and diffusion barrier areas of time membrane which areas are created by physically altering the absorbent membrane. The sample loading device is loaded onto the gel electrophoresis apparatus. A voltage is applied across the gel to establish an electrophoretic field which causes the sample to migrate into the gel and is subsequently scanned for analysis. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. 
     FIG. 1 is a side view of a sample loading device according to the invention; 
     FIG. 2 is a sectional view take along the line  2 — 2  of FIG. 1; 
     FIG. 3 is a sectional view taken along the line  3 — 3  of FIG. 1; 
     FIG. 4 illustrates rotary mechanism for producing a sample loading device of the present invention; 
     FIG. 5 is an enlarged view of part of the mechanism of FIG. 4; 
     FIG. 6 illustrates linear mechanism for producing a sample loading device of the present invention; 
     FIG. 7 shows an alternate linear mechanism for producing a sample loading device; 
     FIG. 8 is a schematic illustration of an RF energy producing mechanism to form a sample device; 
     FIGS. 9A and 9B depict alternative embodiments of sample loading devices containing 64 and 96 sample loading areas, respectively; 
     FIG. 10 is a perspective view of a vertical electrophoretic gel apparatus with a sample loading device according to the present invention; analyzed by polyacrylamide gel electrophoresis from a sample loading device of the present invention and of a sample loading device of the prior art method of using membrane scoring and the addition of hydrophobic ink; and 
     FIG. 12 is a graph comparing the Very High Quality/sample of DNA sequences analyzed by gel electrophoresis from a sample loading device of the present invention and of a sample loading device of the prior art method of using membrane scoring and the addition of hydrophobic ink, as analyzed by Phred software. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Referring to the drawings, where like numerals indicate like elements, there is illustrated in FIGS. 1-3 a sample loading device in accordance with the present invention, generally referred to as  10 . 
     As seen in FIGS. 2 and 3, the sample loading device  10  for an electrophoresis gel is made of a backing material  12 , that carries an absorbent membrane  14  to form a composite material (laminate)  16 . In one embodiment, the backing material  12  has an adhesive  24  for securing the absorbent membrane  14  and the backing material  12  together. 
     This laminate  16  as further discussed below, utilizes the material properties of the absorbent membrane  14  and the backing material  12 . The laminating of the absorbent membrane to the backing material is in contrast to the prior art casting-on of the absorbent to the substrate discussed above. The use of an unsupported absorbent, which can exist without a supporting book as required in the prior art, results in a more robust, adoptable sample loading device  10 . 
     Referring to FIG. 1, the sample loading device  10  has a plurality of sample loading areas or lanes  20  separated from each other by interposed sample diffusion barriers  18 . The diffusion barriers  18  are formed from the same absorbent membrane  14  as the sample loading areas  20 , but the membrane is altered to reduce the absorbent property of the absorbent membrane  14  and create the barrier  18  as explained below. The upper portion of the sample loading device  10  has a non sample holding and labeling area  22 , which use will be described in further detail below. 
     FIG. 2 is a cross sectional view of the sample loading device  10  taken along the line  2 — 2  of FIG.  1 . This view shows the placement and orientation of the backing material  12  and absorbent membrane  14  relative to each other at one of the sample loading areas  20  and also shows the non sample area  22 , which is used for handling and labeling. 
     FIG. 3 is a cross sectional view of the sample loading device  10  taken along the line  3 — 3  of FIG.  1 . The diffusion barrier  18  is compressed/altered absorbent membrane  14  and is secured to the backing material  12 . The compressed/altered absorbent membrane  14  acts as a diffusion barrier  18  which is capable of inhibiting diffusion of the sample placed on the sample loading area  20  of the sample loading device  10 . A sample loading area  20  is shown behind the diffusion barrier  18 . 
     The sample loading device  10  shown in FIGS. 1-3 is representative of an embodiment of the invention and has a plurality of diffusion barriers  18  separating each of the sample loading areas  20 . One such embodiment has a length of 7 inches and a height of 0.75 inches, with 64 sample loading areas  20 . Each of the sample loading areas  20  has a width of 0.089 inches (2.25 mm). Each of the diffusion barriers  18  has a width of 0.035 inches (0.9 mm). The diffusion barrier  18  extends 0.028 inches (7 mm) upward from the bottom edge. 
     The backing material  12  is made of a non-reactive polymer film tape such as a polyamide film tape sold under the trademark Kapton™ tape or a polyester film tape sold under the tradename Mylar™ tape. Both tapes are manufactured by DuPont and sold by Furon/CHR of New Haven, Conn. In a preferred embodiment, the backing material is “Nelatp-581” by Dielectric Polymers, Inc., which is a stable (polyester-like) adhesive backed material preferably. The adhesive is an inert silicone pressure sensitive adhesive. The tape has a width of approximately 0.75-1.00 inches, a thickness (carrier plus adhesive) of between 0.0018 and 0.0026 inches (0.45 and 0.67). 
     The absorbent membrane in a preferred embodiment is any available, DNA transfer membrane, which is an unsupported, neutrally-charged nylon “Biodyne-A Xu/Xu” or Polysulphone absorbent material (Pall Specialty Materials, Port Washington, N.Y.). The absorbent material has a total average thickness of between 0.0100 and 0.0161 inches (0.254 and 0.410 mm) and a pore size of between 0.12 and 0.50 μm. The pore size in a preferred embodiment is reduced by at least 90 percent in the diffusion barriers. 
     The laminate  16 , in a preferred embodiment, is produced by rolling or applying the backing material  12 , which contains adhesive  24 , onto the absorbent membrane  14 . The resultant laminate  16 , as an unformed sample loading device is then subjected to a process which selectively alters the inherent mechanical properties of the absorbent membrane. The process may use pressure, conductive heat, convective heat (such as Laser or RF energy) or a combination of pressure, and heat, or other similar technique to create the sample diffusion barriers  18 , to produce the sample loading device  10  as explained below. The sample diffusion barriers  18  prevent samples from cross contamination and/or diffusion to the adjacent samples on the sample loading device 10. 
     In using mechanical pressure method to form diffusion barriers, the laminate  16  is directed to a forming roller  30  as seen in FIG. 4 that has been fabricated with a predetermined pattern of teeth-like raised areas  32 , which are of such size and spacing to produce the proper size and spacing of the sample diffusion barriers  18  and sample loading areas  20 . As the laminate  16  is fed to the roller  30 , a platen  34  opposes the roller  30  to ensure that the absorbent material layer of the laminate  16  will be compressed against the platen  34  in areas corresponding to the teeth  32  of the roller  30  to form the sample diffusion barrier  18  as best seen in FIG.  5 . The arrows  36  indicate the direction of motion of the roller  30 , the platen  34 , and the laminate  16 . The platen  34  can either be stationary or rotary. In utilizing a stationary platen, the forming roller travels transversely, parallel to the solid (platen) surface  34 . This process alters the material properties of the absorbent membrane  14  to create a pattern of sample diffusion barriers  18 . 
     An alternative to using rotary action to form diffusion barriers by utilizing mechanical pressure is to use a linear motion also utilizing mechanical pressure acting in a plane perpendicular to the laminate surface, as shown in FIG. 6. A stamping device  40  with at least one projection or edge  42 , is attached to a mechanism which moves in a predetermined index linear motion. In one preferred embodiment, the edge  42  is near knife edge in shape and biased by a spring loaded mechanism (not shown). The stamping device  40  as seen in FIG. 6 employs a linear, vertical motion. A stationary platen  44  opposes the stamping device  40 . The laminate  16  is subjected to the pressure between the stamping device  40  and platen  44 , which alters the configuration and material properties of the absorbent membrane to create sample diffusion barriers. The arrows  36  indicate the direction of motion of the stamping device  40 . The arrow  37  indicates the direction of the reciprocating motor of the platen  44 . This operation can be either continuous or non-continuous, depending on the sequence of operations in processing the sample loading device. It is recognized that the laminate material can be fed into the forming device continuously thereby creating a quantity of formed laminate, which is then cut to the desired lengths for sample loading, or individual lengths of laminate material can be formed for sample loading and insertion into the gel electrophoresis apparatus. 
     FIG. 7 shows an alternative embodiment of the stamping device  40  in which there are a plurality of projections  42  which are sized and spaced to produce the proper size and spacing of the sample diffusion barriers  18  and sample loading areas  20 . The stamping device  40  can also produce a horizontal translation motion, in addition to having a vertical motion, to move the laminate  16  to the left as it creates the sample diffusion barriers  18 . The stamping device  40  as it is raised moves to the right prior to engaging the laminate  16  again. 
     It is recognized that the stamping device  40  of FIG. 7 can move in a pure vertical direction with movement of the laminate  16  controlled by other mechanism. Likewise, the stamping device  40  of FIG. 6 likewise can additionally move in a horizontal direction. Other means of applying pressure to the laminate  16  can also be used. The amount of pressure applied to the absorbent membrane to create the diffusion barriers is preferably 2500 psi or greater, and can be in the range of 25,000 to 40,000 psi for a stamping device with  10  projections  42 . 
     In addition to applying pressure, heat in combination with pressure can be used to create diffusion barriers  18 . Less pressure is required to compress the membrane in this embodiment. For example, in one embodiment with the projections  42  heated to approximately 100° F. the amount of pressure needed to create the diffusion barriers can be reduced to 5000 psi for a stamping device with 64 projections  42 . 
     An alternative method to using pressure, or the combination of heat and pressure to create the sample diffusion barrier  18  discussed above, is to apply heat, such as RF energy or laser energy, to the sample loading device to form the sample diffusion barriers  18 . An example of this is illustrated in FIG. 8 When using laser energy or RF energy, the laminate  16  can be attached to or inset on, either temporarily or for the duration of the process, a carrier or surface  52  (also called a platform) that provides a stable and precise platform for the sample diffusion barrier  18  formation process. This platform  52  may be electrically and/or thermally insulating or electrically and/or thermally non-insulating. A laser, RF-generator or similar energy collating, amplifying or focusing device  54  can be positioned and guided so that when exposed to this energy, the material properties of the absorbent membrane  14  are altered to create a pattern of sample diffusion barriers  18 . This operation can either be continuous or non-continuous, depending on the sequence of operations in processing the sample loading device. Other means of applying convection heat in the form of laser energy or RF energy to the absorbent material layer of the laminate can also be used as described above. The amount of laser energy or RF energy applied to the absorbent membrane layer of the laminate to create the diffusion barriers is preferably equivalent to the amount that creates a temperature sufficient to alter the material properties of the absorbent material. 
     An alternate embodiment of the sample loading device  10  is shown in FIGS. 9A and  9 B. These embodiments of a sample loading device  10  can be formed by the methods outlined above. The sample loading device  10  is made such that the sample loading areas  20  do not extend the length of the device  10 . The sample loading device  10  can include a left margin  58  and a right margin  60  which can allow a user to grasp the sample loading device  10  from the side without the danger of disrupting samples placed on the sample loading areas. Additional alignment indicators or nomenclature can be located in these areas. 
     In one embodiment, the sample loading device  10  has 64 sample loading areas  20  as shown in FIG.  9 A. One such example has a length of 7 inches and a height of 0.75 inches. Each of the sample loading areas  20  has a width of 0.089 inches (2.25 mm). Each of the diffusion barriers  18  has a width of 0.035 inches (90 mm). The diffusion barrier  18  extends 0.28 inches (70 mm) upward from the bottom edge. 
     The embodiment shown in FIG. 9B is a sample loading device  10  having 96 sample loading areas  20 . One such example has a length of 7 inches and a height of 0.75 inches. Each of the sample loading areas  20  has a width of 0.089 inches (2.25 mm). Each of the diffusion barriers  18  has a width of 0.035 inches (0.90 mm). The diffusion barrier  18  extends 0.28 inches (7.0 mm) upward from the bottom edge. 
     After formation of the sample loading device  10 , the samples can be added to the sample loading device  10  by, for example, manually spotting the samples with a hand pipette or by automatically spotting the samples, either one at a time or multiple devices, using a robotic workstation. The samples can be any samples known in the-art, including, for example, nucleic acids, proteins and other biologically relevant molecules. 
     The sample loading device  10  can then be inserted into a loading area  68  of a polyacrylamide gel electrophoresis apparatus  70  such as seen in perspective view in FIG.  10 . The apparatus  70  has a well  72  in which a gel  74  is formed or placed. In order to facilitate the loading process, a liquid  76  with a higher viscosity than the buffer solution, such as “Ficoll” can be loaded into the well  72  to a level  78  which just exceeds an upper surface  80  of the gel  74  to further assist in preventing sample diffusion. After the apparatus  70  is filled with the viscous liquid  76 , the sample loading device  10  is positioned within the apparatus  70  so that the sample loading areas  20  are positioned in proximity to the loading area, the top edge,  68  of the gel  74 . 
     The polyacrylamide gel electrophoresis process can be initiated by placing a voltage across the gel via a cathode wire  84  and an anode wire  86  and establishing an electrophoretic field. This process, as described above, forces the samples by their charge to migrate from the sample loading device to the gel. After about five minutes, or when the samples are in the gel, the process can be halted and the sample loading device can be removed. The remaining PEG or glycerol can be washed out of the loading well by flushing with a pipette. The electrophoretic field can then be restarted and the polyacrylamide gel electrophoresis can be allowed to complete its full duration. 
     It is recognized in a preferred embodiment, the sample loading device  10  can have alignment indicators to assist in placing the sample loading device  10  in relation to the polyacrylamide gel electrophoresis apparatus  70 . 
     The device  10  can also have lane marking aids to assist a user in locating particular sample loading lanes  20  in the manually spotting of samples on the device  11 . A stiffening frame can also be utilized if the device  10  is to be handled robotically. 
     EXAMPLES 
     The following examples are for purposes of illustration only, and are not intended to limit the scope of the specification or appended claims. 
     For comparative analysis, a sample loading device according the prior art having both scoring and hydrophobic ink was prepared. Briefly, notches were scored into an absorbent membrane and then were filled with hydrophobic ink. The notches were scored with a push pin, and the hydrophobic ink was applied with a common Bic Round Stic ballpoint pen. The absorbent membrane was supportive nylon. Scoring involves removing absorbent membrane material from the substrate. 
     A sample loading device of the present invention was prepared using the rotary method, as shown in FIG. 4, to convert the absorbent membrane  14  into a sample diffusion barrier. A pressure of about 2500 psi was applied to the absorbent membrane to form the diffusion barriers. 
     To provide an accurate basis for comparison, the sizes and spacings of the prior art sample loading device (comparative comb) and the inventive sample loading device were the same as those discussed above with respect to FIG.  9 B. 
     After preparation as described above, the comparative comb and the sample loading device of the present invention were used in an identical polyacrylamide gel electrophoresis system for comparison. 
     Three 96-well plates of double-stranded DNA templates, for a total of 288 samples, were prepared following the procedure described by Engelstein et al., “Template Preparation for High Throughput Sequencing,”  Microbial and Comparative Genomics , 3(4) (1988), and were sequenced using the Perkin Elmer (PE) Applied Biosystems standard Big  Dye Terminator Cycle Sequencing Ready Reaction Kit , part #4303154, following the 1/4X BigDye Terminator Hydra Sequencing Reactions Protocol. Briefly, a reaction mix was prepared for per 100 reactions that includes 333.33 μl of BigDye mix, 167, μl of BigDye buffer, 447 μl of distilled water and 53 μl of (10 μM) primer. 10 μl of the reaction mixture was then added to a 0.2 ml Micro tube (March Biomedical Products, Rochester, N.Y.). 4 μl of DNA template (50 ng/μl) was then added to each tube. The tubes were then thermocycled following the manufacturer&#39;s instructions. 
     The same DNA template samples were used for the comparative comb and the sample loading device of the present invention. 
     Sequenced samples were precipitated and resuspended, except that the volume of resuspension was 1 μl rather than the standard 2 μl . Briefly, 40 μl of 75% ethanol was added to each sequencing reaction and then centrifuged for 30 minutes at 3000 rpm at 4° C. The plate was then inverted to remove the supernatant, followed by incubation at room temperature to allow the remaining fluid to evaporate. 
     0.5 μl of sample was then manually spotted, using a Rainin pipette, onto each of the comparative comb and the sample loading device of the present invention, where each contained 96 samples. 
     The comparative comb and the sample loading device of the present invention were then inserted into a 0.4 mm well, 0.2 mm 48 cm PE Applied Biosystems gel cassette. Data were collected on an ABI 377 automated sequencer for 10 hours at 2.8 kV. The raw data were transferred to ABI collection software for lane tracking. and initial analysis, and then to Phred for base calling and accuracy assessment. The results obtained from Phred were compared using JMP statistical analysis software. 
     More specifically, the raw data were analyzed by Phred software (Ewing et al., “Base-Calling of Automated Sequencer Traces Using Phred, 1. Accuracy Assessment,”  Genome Research , 8:175-185; and Ewing et al., “Base-Calling of Automated Sequencer Traces Using Phred, 11. Error Probabilities,”  Genome Research , 8:186-194). The results of the Phred analysis were compared using JMP statistical analysis software (JMP 3.2.2 SAS Institute Inc., SAS Campus Drive, Cary, N.C. 27513). 
     From the 288 samples, the comparative comb produced an average readlength/sample of 518 bases/sample with a standard deviation of 313 bases. From the 288 samples, the sample loading device of the present invention produced an average readlength/ sample of 785 bases/sample with a standard deviation of 108 bases. A student&#39;s t test at 95% confidence limits was performed comparing the data. The results of the student&#39;s t test indicated that each data set was significantly different from the other. The results are presented in FIG.  11  and in Table 1 below. 
     
       
         
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                 Number of 
                 Average Readlength 
                 Standard 
                 Standard 
               
               
                 Sample 
                 Samples 
                 in Bases 
                 Deviation 
                 Error Mean 
               
               
                   
               
             
             
               
                 comparative 
                 288 
                 518.462 
                 313.189 
                 18.455 
               
               
                 inventive 
                 288 
                 784.767 
                 108.251 
                  6.379 
               
               
                   
               
             
          
         
       
     
     Phred analysis gives a measure of quality for each of the bases within a sequence call (Ewing et al., “Base-Calling of Automated Sequencer Traces Using Phred, 1. Accuracy Assessment,”  Genome Research , 8:175-185; Ewing et al., “Base-Calling of Automated Sequencer Traces Using Phred, H. Error Probabilities,”  Genome Research , 8:186-194). Phred assigns a number to each nucleotide it calls based on its confidence that the call is correct. The numbers are on a log scale and can range from 0 up. A call of 20 corresponds to a 99% probability that the base call is correct, while a call of 30 corresponds to a 99.9% probability that the base call is correct, while a call of 40 corresponds to a 99.99% probability that the base call is correct, and so on. 
     A term is assigned (i.e., VHQ or Very High Quality data) to base positions with Phred scores greater than or equal to 30 (i.e., 99.9% confidence). The number of VHQ bases is compiled for each read. Looking at the number of VHQ bases achieved for the comparative comb and the sample loading device of the present invention, it can be seen that the sample loading device of the present invention had an average VHQ/sample of 522 bases with a standard deviation of 128 bases. The comparative comb had an average VHQ/sample of 212 bases with a standard deviation of 202 bases. A student&#39;s t test at 95% confidence limits was performed comparing the data. The results of the student&#39;s t test indicated that each data set was significantly different from the other. The results are presented in FIG.  12  and in Table 2 below. 
     
       
         
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
             
             
               
                   
                 Number of 
                   
                 Standard 
                 Standard 
               
               
                 Sample 
                 Samples 
                 VHQ Bases 
                 Deviation 
                 Error Mean 
               
               
                 comparative 
                 288 
                 212.403 
                 202.553 
                 11.936 
               
               
                 inventive 
                 288 
                 521.583 
                 128.669 
                  7.582 
               
               
                   
               
             
          
         
       
     
     Based on the above results it can be seen that the sample loading device of the present invention achieved readlengths of 267 more bases than the comparative comb. These data show that the sample loading device of the present invention had a 51% increase in readlength over the comparative comb. Also, the sample loading device of the present invention achieved 310 more VHQ bases than the comparative comb. These data show that the sample loading device of the present invention had an increase of over 146% in the accuracy of the bases called. It is clear from these data that the sample loading device of the present invention produced results that were unexpectedly superior to the results produced by the comparative comb. 
     Each of the patent applications and publications cited in the present specification is hereby incorporated by reference herein in its entirety. 
     While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.