Abstract:
DNA is detected using complementary RNA probes and an enzyme that attacks and hydrolyzes the RNA probes only when it has hybridized with target DNA. A low concentration of target DNA can therefore successively hydrolyze a larger amount of RNA whose loss may then be detected to indirectly determine the presence of the target DNA.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
     BACKGROUND OF THE INVENTION  
       [0001]     The present invention relates to instruments and methods for the detection and/or identification of DNA.  
         [0002]     The detection and identification of oligonucleotides, genomic and PCR (polymerase chain reaction) amplified DNA is important in a wide variety of applications ranging from basic research and medical diagnostics to the detection of bioterrorism. One commonly used method for the detection and identification of DNA makes use of “gene chips”, in which ssDNA molecules are immobilized onto a substrate. These probe molecules may be arranged at the intersections of a rectangular grid over the surface of the substrate, each with the ability to hybridize to their complementary target DNA sequence. For most applications the DNA target of interest is PCR amplified to increase the amount of DNA to be detected. The most common detection technique with these systems is fluorescence, so the target DNA must be fluorescently tagged before it is flowed over the surface of the gene chip for detection. By viewing concentrated fluorescence signal caused by the hybridization of the target DNA to particular probes on the gene chip, and by knowing the nucleotide sequence of the probe molecules at those points of attachment, the presence of particular target molecules and their composition may be determined. These types of gene chips, which rely on fluorescence for the detection of complementary DNA sequences, are commercially available from companies such as Affymetrix and are widely used in the field of biotechnology. Other tagging methods including the use of radioactive substances may also be used, but is done so less frequently.  
         [0003]     A promising technique that eliminates the need to use fluorescent or enzymatic tags is surface plasmon resonance (SPR). In SPR, a thin metallic film is illuminated from the backside “reflecting side” of the film. At a certain angle known as the plasmon angle, the energy from the illumination is coupled into electromagnetic waves creating a resonant condition (surface plasmon resonance) that is highly sensitive to surface conditions on the “sensing side” of the film opposite to the reflecting side.  
         [0004]     SPR can be used in many different modes, such as SPR imaging, which allows multiple probe spots to be analyzed in parallel, so that arrays of different DNA probes may be attached to the surface. In the technique of SPR imaging, p-polarized light impinges on a prism, gold thin film, and flow cell assembly at a fixed angle. The reflected light is passed through a narrow band pass filter and collected by a CCD camera. In this technique probe molecules are covalently linked to discrete positions on the sensing side of the film to selectively bind with target molecules in the solution to be analyzed. The binding of the targets to the surface bound probes causes localized changes in the index of refraction at those spots, which are detected by the CCD camera. This data can then be used to identify the presence and composition of the target molecules by the location of the detected binding event.  
         [0005]     While SPR imaging may eliminate the need for tagging target molecules, current SPR imaging methods can only detect DNA target molecules down to concentrations of approximately 10 nM. Accordingly time consuming and cumbersome amplification techniques such as PCR must be employed when looking at very low DNA copy numbers.  
       SUMMARY OF THE INVENTION  
       [0006]     The present invention provides a way to boost the sensitivity of SPR or other DNA detection techniques by as much as 10,000 times. The invention raises the possibility of direct detection of genomic DNA without the need for target amplification using PCR or the like.  
         [0007]     Generally, the invention employs a mechanism for selectively destroying RNA probe molecules on a test surface when the RNA molecules have hybridized with target DNA in the sample being tested. When the RNA is destroyed, the previously hybridized DNA molecules may hybridize with new complementary RNA probe molecules initiating further cycles of destruction of complementary RNA probes. The ability of a single target DNA molecule to initiate the destruction of many complementary RNA probe molecules allows the detection of very low concentrations of DNA by observing RNA loss. This “intensification” effect, which does not require tagging or increasing the number of DNA target molecules when used with SPR imaging, can greatly simplify the detection of small concentrations of DNA.  
         [0008]     Specifically then, the present invention provides a method of detecting target DNA molecules comprising the steps of introducing target DNA molecules into a test element holding RNA molecules complementary to the target DNA molecules and selectively destroying the RNA molecules that have hybridized with the target DNA molecules by an enzymatic process. The loss of RNA molecules caused by target DNA molecules successively hybridizing with different RNA molecules and their subsequent destruction by an enzymatic process is then detected.  
         [0009]     It is one object of the invention to provide direct detection of extremely low concentrations of DNA.  
         [0010]     The destruction of the RNA may be performed by the enzyme RNase H.  
         [0011]     It is another object of the invention to provide a simple method for selectively destroying hybridized RNA suitable for use with the present invention.  
         [0012]     The enzyme may be introduced into the test cell at the same time as the target DNA in single a solution.  
         [0013]     Thus it is another object of the invention to provide for a convenient method of manipulating the necessary test sample and enzyme.  
         [0014]     The test element may be a surface to which RNA molecules are attached.  
         [0015]     Thus it is another object of the invention to provide a structure that may spatially support and segregate multiple different RNA molecules to provide for parallel analyses of DNA molecules comparable to that provided by conventional gene chips.  
         [0016]     The surface may be selected from the group consisting of: diamond, glass, silicon, and gold.  
         [0017]     Thus it is another object of the invention to provide a technique adaptable to a wide variety of detection technologies including SPR, fluorescent microscopy, and direct electrical detection.  
         [0018]     The test element may be a surface plasmon resonance test cell, which consists of a microarray fabricated on a chemically modified gold surface and a flow cell, and the detection may be performed by an SPR imaging instrument for detecting the loss of RNA molecules from the test cell.  
         [0019]     Thus it is one object of the invention to provide a system suitable for use with SPR instruments. It is another object of the invention to provide a detection system that avoids the need to tag the sample DNA.  
         [0020]     Alternatively, the RNA molecules may be tagged with a fluorescent material and the detection step may be performed with a florescence detection apparatus, such as a fluorescence microscope or a fluoroimager.  
         [0021]     It is thus another object of the invention to provide an intensification system applicable to a wide range of detection techniques.  
         [0022]     These particular objects and advantages may apply to only some embodiments falling within the claims and thus do not define the scope of the invention. 
     
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0023]      FIG. 1  is a block diagram of a surface plasmon resonance imaging device suitable for use with the present invention, showing a test element allowing for the flow of target DNA and enzymes over a surface having attached RNA probes;  
         [0024]      FIG. 2  is a plot showing a relationship between reflectance and incident angle in the SPR instrument for two concentrations of RNA probes at a particular site;  
         [0025]      FIG. 3  is an elevational diagram showing the RNA probes attached to the substrate as may hybridize with a single stranded DNA target in solution with the enzyme RNase H;  
         [0026]      FIG. 4  is a figure similar to that of  FIG. 3  showing the destruction of a first hybridized RNA probe by RNase H, freeing the DNA for subsequent hybridization;  
         [0027]      FIG. 5  is a figure similar to that of  FIGS. 3 and 4  showing the destruction of a second hybridized RNA probe by RNase H, again freeing the DNA for subsequent hybridization;  
         [0028]      FIG. 6  is a figure similar to that of  FIGS. 3-5  showing completion of the destruction of the RNA probes on the test surface;  
         [0029]      FIG. 7  is an elevational diagram of an alternative test surface having two different RNA probes, one complementary and one not complementary to a target DNA, and a DNA probe complementary to a target DNA;  
         [0030]      FIGS. 8   a - 8   c  show plan images of the test surface of  FIG. 7  at successive stages of hybridization and RNA hydrolysis;  
         [0031]      FIG. 9  is a flow chart of the method of the present invention;  
         [0032]      FIG. 10  is a planar view of a substrate providing probe molecules arranged in asymmetrical tile patterns for rotational identification;  
         [0033]      FIG. 11  is a simplified representation of an alternative detection system using fluorescence sensitive scanning; and  
         [0034]      FIG. 12  is a figure similar to  FIG. 12  of an alternative detection system using radioactively tagged probes. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0035]     Referring now to  FIG. 1 , a surface plasmon resonance (SPR) device  10  may include a test element  12  providing a chamber  14 . One wall of the chamber  14  provides a transparent test substrate  16  having on its inner surface, facing the chamber  14 , a gold film  18 . A front surface of the gold film  18  facing the chamber  14  may be spotted at probe locations  20  with molecules that may include RNA probe and control molecules and DNA control molecules in a regular pattern as will be described below.  
         [0036]     In the SPR device, a collimated white light source  22 , directs lights through a polarizer  24  at an angle q through a coupling prism to strike the rear surface of the gold film  18  after passing through the substrate  16 . The light reflects off the gold film  18  at equal and opposite angle q′ to be received through a narrow band pass filter  26  by a CCD camera  28 . The CCD camera  28  is focused on the rear surface of the gold film  18  to provide an image of the reflected light from that surface. As is understood in the art, the amount of material on the inner surface of the gold film  18  will affect the amount of reflected light received by the CCD camera  28  and thus, the image produced by the CCD camera  28 , may be used to detect the amount of material on the front surface of the gold film  18 . Systems suitable for this purpose are described in co-pending U.S. patent applications Ser. No. 10/411,583 filed Apr. 10, 2003, and Ser. No. 10/602,243 filed Jun. 24, 2003, assigned to the assignee of the present invention and hereby incorporated by reference.  
         [0037]     The chamber  14  includes an inlet port  30  which may be connected by means of valves  32  to different fluid flow lines providing liquids for washing the surface of the front of gold film  18  and for introducing DNA samples and an enzyme into the chamber  14  as will be described. An exit port  34  allows liquids to be withdrawn from the chamber  14  and or recirculated.  
         [0038]     Referring now to  FIG. 2 , reflectance  36  off the rear surface of the gold film  18  is a function of the angles q and q′ which may be adjusted to a fixed angle in order to produce a baseline reflectance  38  from the gold film  18  delineating the probe molecules at the probe locations  20  prior to reaction with sample DNA. A loss of material from the probe locations  20  will cause the reflectance curve to move to  36 ′ causing a significantly decreased reflectance at the fixed angle indicated by level  38 ′.  
         [0039]     Referring now to  FIGS. 3 and 9  per process block  42 , RNA molecules  40  having a sequence of nucleotides complementary to an intended target DNA molecule may be covalently attached at predetermined probe locations  20  to the front surface of the gold film  18  to create an array. The RNA molecules  40  at each probe location  20  will have the same nucleotide sequence. In addition, and referring to  FIG. 7 , control molecules  46  being either RNA or DNA may be covalently attached to other predetermined probe locations  20  on the front surface of the gold film  18 . The creation of the array on the gold film  18  containing RNA molecules  40  and the control molecules  46  and may use protection/de-protection surface chemistry and photo patterning adapted from Brockman, et al. and well known in the art. See generally, Brockman, J. M.; Frutos, A. G.; Corn, R. M.; J. Am. Chem. Soc. 1999, 121 8044-8051.  
         [0040]     Referring momentarily to  FIG. 10 , the probe locations  20  may be square areas laid out in rectilinear columns and rows over the surface of the gold film  18 . These probe locations  20  may be collected into zones  48   a ,  48   b , and  48   c , each having a common RNA molecule  40  or control molecule  46  so as to react similarly. The shapes of the zones  48   a ,  48   b , and  48   c  are selected so as to be rotationally asymmetric so that the rotational orientation of the substrate  16  may be determined unambiguously by observation of the zones  48   a - 48   c  or any individual zone  48   a - 48   c.    
         [0041]     In an example embodiment, the probe locations  20  of zone  48   a  have RNA molecules  40  that will hybridize with an expected target DNA, while the probe locations  20  of zone  48   b  having RNA molecules  40 ′ that will not hybridize with the expected target DNA. The probe locations  20  of zone  48   c , in contrast, may have DNA molecules that will hybridize with the target DNA. These latter two zones  48   b  and  48   c  provide control and calibration zones as will be described below. Alternatively, each of the zones  48   a - 48   c  may have RNA molecules  40  that will hybridize with different expected target DNA. The number of zones  48  and probe locations  20  in a zone may be freely varied.  
         [0042]     Referring again to  FIGS. 3 and 9 , once the array is finished per process block  42 , the slide is then mounted into the SPR imager and the front surface of the gold film  18  is rinsed per process block  44 . All of the solutions used in the following steps may be autoclaved for sterilization. The rinsing process begins with water being washed over the gold film  18  to rinse away anything absorbed to the surface. Referring to  FIG. 1 , the water may be introduced through one of the valves  32 . A buffer solution is then washed over the front surface of the gold film  18  made up of, for example, 50 mM Tris (pH 8.3), 50 mM KCl, and 10 mM MgCl2, 0.5 mM spermidine, and 10 mM DTT.  
         [0043]     Referring still to  FIG. 9 , after the rinsing at process block  50 , a first image of the gold film surface  18 , using the SPR imaging instrument  10 , may then be acquired. In this image, different probe locations  20  will have different reflectance and this reflectance may be measured and captured as a digital value related to the pixels associated with that portion of the image.  
         [0044]     Next, at process block  52 , a solution of 1 pM of the target DNA in 500 uL of the same buffer solution may rinsed over the gold film  18  introduced via one of the valves  32  shown in  FIG. 1 . This may be followed by the introduction of RNase H per process block  56 , or per the preferred embodiment, 30 units (0.5 uL) of RNase H may be added to the 500 uL solution of DNA previously described so that the target DNA and enzyme can be present at the surface simultaneously. This process is not limited to only photopatterned arrays or large volume cells but may also make us of other techniques such as microfluidics.  
         [0045]     Referring to  FIG. 3  at this time, a target DNA molecule  54  may hybridize with one of the complementary RNA molecules  40   c  at a probe location  20 .  
         [0046]     Referring to  FIG. 4 , the RNase H  58  is an enzyme that has the property of hydrolyzing RNA molecules  40  only when an RNA molecule  40  is hybridized with a target DNA molecule  54 . Thus as shown in  FIG. 4 , The RNase H  58  attacks the RNA  40   c  previously bonded to target DNA molecule  54 , hydrolyzing the RNA molecules  40   c  and leaving the target DNA molecule  54  unharmed, free to react again with another RNA molecule  40 . As also shown in  FIG. 4 , typically during the hydrolysis of one RNA molecule  40   c , another DNA molecule  54  may be reacting with a different RNA molecule  40 .  
         [0047]     Referring now to  FIG. 5 , the DNA molecule  54  released in  FIG. 4  after the hydrolysis of RNA molecule  40   c , may hybridize with another RNA molecule  40   d  attached to the gold surface  18  while the RNA molecules  40   a  of  FIG. 4 , hybridized to target DNA molecule  54 , is attacked by RNase H  58  to release its target DNA molecule  54  for subsequent hybridization.  
         [0048]     As shown in  FIG. 6 , ultimately all of the RNA molecule  40   a - 40   d  complementary to the target DNA molecule  54  may be hydrolyzed and thus removed from the gold surface  18  in the given probe region  20 . Because a single DNA molecule  54  may hydrolyze multiple RNA molecules  40 , the effect of even a few DNA molecules is intensified. As an enzyme, the RNase H  58  is not consumed during the hydrolysis process. The present invention can provide a 10,000 times increase in sensitivity through this intensification process.  
         [0049]     The solution of DNA molecules  54  and RNase H  58  is allowed to sit on the gold surface  18  for approximately thirty minutes to allow repeated hybridization and enzymatic hydrolysis of the RNA molecules  40  attached on the gold surface  18 .  
         [0050]     Referring again to  FIG. 9 , at the conclusion of the hydrolyzation process of process blocks  52  and  56  described above, the gold surface  18  is washed again with buffer, and as previously described, the washing as indicated by process block  59 .  
         [0051]     At succeeding process block  60 , a second image of the gold film  18  is taken using the SPR instrument  10 . In this image, those probe locations  20  where hydrolyses of RNA molecules will have different reflectance from the same probe locations in the image captured at previous process block  50 . The reflectance of these probe locations are also captured digital values.  
         [0052]     The arithmetic difference between the reflectance of given probe locations  20  may be determined by a computer receiving the two images according to techniques well known in the art as indicated by process block  62 . Per process block  64 , a threshold may be applied to the difference signal to determine which probe locations  20  have had reactions that have destroyed their RNA molecules  40  and thus which have encountered specific target DNA molecules  54 . Experiments have determined that a perceptible difference in reflectance will occur for DNA target molecules in solution at concentrations down to 1 pM.  
         [0053]     The output of this result may be indicated to the user through a subtraction image or an automated image processing system of types well known in the art as indicated by process block  66 .  
         [0054]     Referring now to  FIG. 7 , the gold surface  18  may include not only RNA molecules  40  intended to hybridize with target DNA molecules  54 , but also RNA molecules  40 ′ intentionally of a sequence not binding with expected target DNA molecules  54  and DNA molecules  41  expected to hybridize with expected target DNA  54  which also hybridize with at least one of the RNA molecules  40 . These RNA molecules  40 ′ and DNA molecules  41  serve as controls for monitoring the process, for example, to make sure the substrate is viable, and to provide a reference against which automatic measurement thresholds may be established.  
         [0055]     For a gold surface  18  with control materials, the SPR instrument  10  is initially adjusted so that the reflectance of the gold surface  18  at the time of image of process block  50  of  FIG. 9  is somewhere just off the SPR angle of curve  36  of  FIG. 2  to provide a baseline reflectance  38 .  
         [0056]     Referring now to  FIG. 8   a , at the time of introduction of sample DNA molecules  54  of process block  52  of  FIG. 9 , the probe locations  20   a  and  20   c  will show increased reflectance caused by a binding of target DNA molecules  54  to the RNA molecules  40  and DNA molecules  41 .  
         [0057]     Referring to  FIG. 8   b , at the time of introduction of the RNase H  58  of process block  56  of  FIG. 9 , the probe location  20   a  will show decreased reflectance caused by a hydrolysis of the RNA molecules  40  while probe location  20   c  will not show this change.  
         [0058]     At the time of the taking of a second image per process block  60  of  FIG. 9 , the reflectance of probe locations  20   a  will have dropped considerably below the reflectance of probe locations  20   b  and probe locations  20   c  caused by the loss of RNA molecules  40  in that probe location  20   a.  The reflectance of probe locations  20   b  or  20   c  may be used to establish a threshold used in process block  64  of  FIG. 9  as described above.  
         [0059]     While the present invention works well with SPR devices  10 , which eliminates the need to tag the DNA molecules  54 , the invention also may find application in a wide variety of other detection systems including, for example, those which detect the presence of the RNA probes  40  directly through electrical interaction with a treated silicon substrate.  
         [0060]     The present invention also allows conventional fluorescent gene chip techniques to be used while still avoiding the need to tag the target DNA molecules  54 . Referring to  FIG. 11 , in this case, the RNA molecules  40  may be tagged with a fluorescent dye  68 . A conventional fluorescent scanner  70  of a type known in the art, may then stimulate the fluorescent dye  68  and record an image structurally similar to the SPR image at step  50  of  FIG. 9 . Upon completion of the hydrolysis, a second image may be obtained per block  60  of  FIG. 9  and a subtraction image based on fluorescence rather than reflectance may be made to determine the loss of RNA molecules  40  caused by the hydrolysis process.  
         [0061]     Referring to  FIG. 12 , alternatively, the RNA molecules  40  may be tagged with a radioactive material  70  and an image obtained by photographic film  72  or other techniques well known in the art.  
         [0062]     Other techniques for detection of the loss of RNA molecules may also be used, and therefore, it will be understood that a wide variety of substrate materials may be used including glass and diamond.  
         [0063]     It is specifically intended that the present invention not be limited to the embodiments and illustrations contained herein, but include modified forms of those embodiments including portions of the embodiments and combinations of elements of different embodiments as come within the scope of the following claims.