Patent Publication Number: US-2018052106-A1

Title: Dual detection scheme for dna sequencing

Description:
FIELD OF THE INVENTION 
     This invention generally relates to DNA sequencing and more specifically to fluorescent and chemical sensing of nucleotide incorporation events. 
     BACKGROUND OF THE INVENTION 
     To carry out the sequencing of the human genome, the DNA (deoxyribonucleic acid) is cut into short fragments, the fragments are sequenced simultaneously and the data may then be assembled using sophisticated computer technology. DNA sequencing is the process of determining the precise order of nucleotides (thymine, adenine, guanine, and cytosine) within a DNA molecule. DNA sequencing by synthesis is commonly achieved using one of two sensor modalities to monitor nucleotide incorporation. The two sensed modes or modalities are optical detection of fluorescently tagged nucleotides and the use of ion selective field effect transistors (ISFETs) to detect hydrogen ions that are released when a nucleotide is incorporated onto a target DNA fragment. 
     Typically, the optical detection schemes incorporate complicated optical instrumentation to scan across large substrates. The four nucleotides are distinguished by assigning a different wavelength fluorophore to each nucleotide. By assigning different target DNA fragments to each site on a substrate and monitoring fluorescence color, the identity of each nucleotide that incorporates onto the target fragment may be determined. This system has the advantage that all nucleotides may be introduced simultaneously, but requires a complex optical system to monitor four colors simultaneously across a large substrate. This system also requires the use of modified polymerases that have been selectively engineered to accommodate the industry standard dual-modified nucleotides. The dual-modified nucleotides are independently tagged with a fluorescent moiety and a 3′-block to prevent subsequent nucleotide polymerization. 
     Alternatively, the incorporation of nucleotides onto the target fragment may be determined by monitoring a local pH change that occurs as hydrogen ions are released during a nucleotide base incorporation event. Typically, target DNA fragments are distributed onto beads and biologically amplified on the beads using PCR (Polymerase Chain Reaction). The beads are then loaded onto an array of ISFETs such that one bead is incorporated into one well on top of each ISFET. The four nucleotides, one at a time, are then flowed across the array in serial fashion, and the pH change upon nucleotide incorporation is monitored at each pixel (each ISFET of the array) to determine to which target or pixel a nucleotide base has incorporated. This system has the advantage that it does not require a complex optical system to monitor the incorporation, but conversely the nucleotides must be flowed serially across the ISFET array and there are issues in discerning homopolymer regions (i.e., a region in the target DNA fragments or strands with a number of the same bases occurring in a row). Another advantage is that standard polymerases can be used in the sequencing reaction since standard nucleotides are used for this scheme. 
     In order to simplify the optical system associated with the fluorescent detection process, schemes have been proposed which only use a single color fluorescence for detection of all four nucleotides. The most recently announced one-channel chemistry scheme (from Illumina) describes the following steps: “thymine will have a permanent fluorescent label. Adenine will have the same fluorescent label, but that dye will be removable. Guanine will be permanently dark. And, cytosine will start dark but will be tagged so that a dye can be added to it.” Illumina has then described how this scheme would work to read the DNA. “Essentially, in a first image of the four nucleotides, A and T are both labeled and detectable. Then, in the second image, the dye is cleaved from A and added to C. In the second image, only C and T fluoresce. By combining the information from the two images, all four bases are easily discriminated.” 
     The issue with this scheme for clinical applications is that the incorporation of the nucleotide guanine is a null event. That is, the site will be dark if there is a guanine incorporation event, or if there is no incorporation at all. This potentially introduces errors into the detected DNA sequences and is unlikely to receive FDA approval. 
     It would be highly advantageous, therefore, to remedy the foregoing and other deficiencies inherent in the prior art. 
     Accordingly, it is an object of the present invention to provide a new and improved detection process for DNA sequencing. 
     It is another object of the present invention to provide a new and improved detection process for DNA sequencing incorporating both an optical detection process and a process of detecting hydrogen ions that are released when a nucleotide is incorporated onto a target DNA fragment. 
     SUMMARY OF THE INVENTION 
     The desired objects of the instant invention are achieved in accordance with apparatus for fluorescent and ion sensing of DNA nucleotide incorporation events including DNA nucleotide incorporation structure designed to have sequencing primers bonded to a surface for the incorporation of DNA nucleotides thereon, with at least some of the DNA nucleotides having a fluorescent label. A photodiode is positioned adjacent to the incorporation structure and an illumination device positioned in proximity to the DNA nucleotide incorporation structure to illuminate DNA nucleotides incorporated onto the sequencing primers. The illumination device excites the fluorescent labels when incorporation occurs and the photodiode is positioned to sense the fluorescence from the excited labels. Ion sensing apparatus is additionally positioned adjacent to the DNA nucleotide incorporation structure including a metal oxide thin film transistor with a gate electrically coupled to receive an electrical signal indicative of ion emissions produced by the DNA nucleotide incorporated onto DNA target fragments or sequencing primers. 
     The desired objects of the instant invention are also achieved in accordance with a method of fabricating apparatus for deoxyribonucleic acid (DNA) sequencing and more specifically for fluorescent and ion sensing of DNA nucleotide incorporation events. The method includes the steps of providing a substrate, fabricating either ion sensing apparatus including a metal oxide thin film transistor or an amorphous silicon photodiode on the substrate, and fabricating the other of the ion sensing apparatus and the amorphous silicon photodiode adjacent to the one fabricated on the substrate. The method also includes fabricating either a reservoir or a well overlying the amorphous silicon photodiode, and fabricating both the reservoir and the well with a transparent bottom and a sensing layer incorporated in the bottom. The sensing layer includes an ion sensing element positioned to sense ion emissions in the reservoir or the well and electrically coupling the sensing element to a gate of the metal oxide thin film transistor. Both the reservoir and the well are designed to have DNA target fragments or sequencing primers bonded to a surface for the incorporation of DNA nucleotides onto the DNA target fragments or sequencing primers, at least some of the DNA nucleotides having a fluorescent label. The method also includes a step of providing an illumination device positioned adjacent the reservoir or the well to illuminate DNA nucleotides incorporated onto the DNA target fragments or sequencing primers, the illumination device exciting the fluorescent labels when incorporation occurs with the photodiode positioned to sense the excited fluorescent labels. 
     The desired objects of the instant invention are also achieved in accordance with a method of deoxyribonucleic acid (DNA) sequencing and more specifically fluorescent and ion sensing of DNA nucleotide incorporation events. The method includes the steps of: providing a sensing pad and bonding sequencing primers to a surface of the sensing pad; attaching target DNA fragments to the sequencing primers; attaching sequencing polymerase enzymes to the target DNA fragments; using the sequencing polymerase enzymes, incorporating matching nucleotides with the sequencing primers, whereas hydrogen ions are released upon incorporation of the matching nucleotides; attaching blocking molecules to the matching nucleotides and labeling the matching nucleotides with fluorophores; illuminating the attached and labeled target DNA fragments and sequencing primers to excite the fluorophores; sensing the release of hydrogen ions and the fluorescent emission of the fluorophores; cleaving the blocking molecules and the matching nucleotides from the sequencing primers; and repeating the steps of using, attaching blocking molecules, illuminating and sensing the release of hydrogen ions and the sensing of the fluorescence for additional sequencing events. 
     The desired objects of the instant invention are further achieved in accordance with a preferred embodiment of the above method wherein the steps of providing the sensing pad and sensing the release of hydrogen ions and the excitation of the fluorophores include providing apparatus for sensing both fluorescent and ion emissions during nucleotide incorporation events, the apparatus including an ion sensing metal oxide thin film transistor and an amorphous silicon photodiode on a common substrate, and one of a reservoir and a well overlying the amorphous silicon photodiode, both the reservoir and the well having a transparent bottom and a sensing layer incorporated in the bottom, the sensing layer including an ion sensing element positioned to sense ion emissions in the reservoir or the well, the sensing element electrically coupled to a gate of the metal oxide thin film transistor, and both the reservoir and the well having a surface that forms the sensing pad. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The foregoing and further and more specific objects and advantages of the instant invention will become readily apparent to those skilled in the art from the following detailed description of a preferred embodiment thereof taken in conjunction with the drawings, in which: 
         FIG. 1  is a simplified layer diagram illustrating a combined MOTFT ion sensitive and optical detection structure in accordance with the present invention; 
         FIG. 2  is a simplified layer diagram illustrating a combined MOTFT ion sensitive and optical detection structure with an a-Si photodiode on top of the ion sensitive MOTFT in accordance with the present invention; 
         FIG. 3  illustrates sequencing primers bound to a sensing pad surface, such as the sensing pad of structure  60 ; 
         FIG. 4  illustrates sequencing primers bound to a bead surface, such as the bead illustrated in the well of structure  10 ; 
         FIGS. 5 through 12  illustrate steps in a chemical process for improving detection of nucleotide incorporation; 
         FIG. 13  illustrates one photocleaving and illumination structure; 
         FIG. 14  illustrates another photocleaving and illumination structure; and 
         FIG. 15  illustrates the UV LED wavelength versus the green LED wavelength to show the separation. 
     
    
    
     DETAILED DESCRIPTION OF THE DRAWINGS 
     Turning to  FIG. 1 , a structure  10  is illustrated which includes and combines an ion sensitive MOTFT (metal oxide thin film transistor)  12  and an optical detection thin film photodiode  14 . Structure  10  is fabricated on a substrate  16  which in this preferred embodiment is glass but could be other transparent material, such as plastic or the like. A lower contact  17  for photodiode  14  and structure  10  is deposited on the upper surface of substrate  16  and defines an area on which the combined MOTFT  12  and photodiode  14  are fabricated. Contact  17  may be any of the well-known conductive materials used in the semiconductor industry, such as indium-tin-oxide (ITO), Mo, Al, and the like. An n+ doped layer  18  of a-Si (amorphous silicon) is deposited on the upper surface of contact  17  and an intrinsic or insulating layer  19  of a-Si is deposited on the upper surface of layer  18 , both of which extend substantially over the area or upper surface of contact  17 . A bond pad  20  is positioned on the upper surface of substrate  16  to one side so as to be in an area not covered by contact  17  but which is readily accessible, along with an edge of contact  17 , for easy interrogation of MOTFT  12  and photodiode  14 , preferably, simultaneously or in a specific order on a single incorporation event. 
     A p+ doped layer  23  of a-Si is deposited on the upper surface of layer  19  adjacent an edge of layer  19  in a smaller area and forms amorphous silicon thin film diode  14  in combination with layers  19  and  18 . A layer  25  of transparent conductive material, such as ITO, is deposited on the upper surface of layer  23  and provides an upper contact for photodiode  14 . As will be explained in more detail below, photodiode  14  is positioned relative to MOTFT  12  so that a well can be formed adjacent to MOTFT  12  directly above and in light communication with photodiode  14 . 
     A layer  26  of silicon nitride (SiN) is formed or deposited so as to extend over the entire area of layer  19 , including the area covered by layer  23  and layer  25  overlying layer  23 . A layer  27  of transparent conductive material (e.g. ITO) is deposited on the upper surface of SiN layer  26  so as to extend from an area above layer  25  to a mid-point in structure  10  where it serves as a bottom gate for MOTFT  12 . A layer  30  of gate dielectric material, preferably a second layer of SiN, is deposited or formed over a portion of conductive layer  27  in the central area of structure  10 . A layer  32  of semiconductor metal oxide is deposited/formed on the upper surface of gate dielectric layer  30  overlying a portion of bottom gate layer  27 . Spaced apart source/drain contacts  34  are deposited/formed on semiconductor metal oxide layer  32 . Optionally, an additional layer  36  of gate dielectric material is deposited/formed on the upper surface of semiconductor metal oxide layer  32  between source/drain contacts  34  and a metal top gate  38  is deposited/formed on the upper surface of gate dielectric layer  36  so as to define a channel area in semiconductor metal oxide layer  32 . As will be understood, source/drain contacts  34  and top gate  38  include electrical connections (not shown) designed to electrically couple MOTFT  12  into external circuitry, such as a switch matrix or the like, and to bond pad  20 . Also, lower gate layer  27  is coupled to conductive layer  25  so as to couple photodiode  14  into the circuit. 
     A thick layer  40  of insulating encapsulation material is deposited over MOTFT  12  and photodiode  14 . As will be understood by artisans in the field, the insulating encapsulation material is selected to have a minimum and preferably no effect on both the electrical and chemical components of structure  10  and the subject tests. A well  42  is formed in layer  40  in overlying relationship with photodiode  14  and more specifically p+ doped a-Si layer  23 . The horizontal extent of well  42  is slightly greater than the extent of p+ doped a-Si layer  23  and extends vertically into layer  40  to conductive layer  27  (which might operate for example as an etch-stop). A layer  44  of dielectric or insulating material is deposited in the bottom of well  42  to electrically insulate well  42  from conductive layer  27 . All of the material between well  42  and photodiode  14  is generally referred to as the ‘bottom’ of well  42  for convenience. The overall size (depth and width) of well  42  is designed to receive therein a bead  46  with biologically amplified target DNA fragments distributed thereon. A fluid  48  is used to carry nucleotides serially into well  42  for testing purposes. 
     In operation, when a labeled nucleotide carried by liquid  48  into well  42  is incorporated into the target DNA fragments on bead  46 , a fluorescence event will occur when bead  42  is illuminated by an illumination source  49 . The presence or absence of fluorescence is sensed by photodiode  14  which appears as a signal on contact  17 . Simultaneously, the incorporation of nucleotides onto the target fragment release hydrogen ions and produce a change in the pH of liquid  48  in well  42 . The change in pH is sensed by a small change in voltage on conductive layer  27  connected to the bottom gate of MOTFT  12 . The small change in voltage on the bottom gate acts similar to a bias so that a larger signal on the top gate is required to activate (i.e. turn ON or turn OFF) MOTFT  12 . Thus, the small signal is essentially amplified which, depending upon the design and construction of MOTFT  12 , can be as much as a factor of  10 . As is well-known in the art, the degree of such charge amplification is determined by the relative capacitances of the top and bottom gates. Here it should be understood that through proper design and selection of materials, MOTFT  12  can be fabricated with extremely low leakage current and enhanced mobility of the channel. These characteristics allow the use of MOTFT  12  as a sensor of the small signals generated by the change in pH as well as convenient incorporation into a matrix of structures  10 , if desired. Many examples of designs and materials for MOTFT  12  can be found in, for example, U.S. Pat. No. 7,812,346, entitled “Metal Oxide TFT with Improved Carrier Mobility”, issued Oct. 12, 2010; U.S. Pat. No. 7,977,151, entitled “Double Self Aligned Metal Oxide TFT”, issued Jul. 12, 2011; and U.S. Pat. No. 8,679,905, entitled “Metal Oxide TFT with Improved Source/Drain Contacts”, issued Mar. 25, 2014, all of which are incorporated herein by reference. 
     Turning to  FIG. 2 , another example of a combined MOTFT ion sensitive and optical detection structure  60  is illustrated which includes and combines an ion sensitive MOTFT (metal oxide thin film transistor)  62  and an optical detection thin film photodiode  64 . Structure  60  is fabricated on a substrate  66  which in this preferred embodiment is glass but could be other materials not necessarily transparent (unless required by the fabrication of MOTFT  62 ). Gate metal  68  is deposited on the surface of substrate  66  so as to extend from a central portion of structure  60 , where it serves as a bottom gate for MOTFT  62 , to adjacent the right-hand edge of substrate  66 . A layer  69  of gate dielectric, which in this preferred embodiment is SiN but may be other insulating material, is deposited over gate metal  68 . An active layer  70  of semiconductive metal oxide is deposited in overlying relationship to a bottom gate portion of gate metal  68 . Source/drain contacts  72  are formed in spaced apart relationship in contact with the upper surface of active layer  70 . A layer  74  of gate dielectric or insulating material is deposited on the upper surface of active layer  70  between source/drain contacts  72  and gate metal is deposited on the upper surface of gate dielectric layer  74  to define a channel in active layer  70 . As will be understood, source/drain contacts  72  and top gate  76  include electrical connections (not shown) designed to electrically couple MOTFT  62  into external circuitry, such as a matrix or the like. 
     A thick layer  80  of insulating encapsulation material is deposited over MOTFT  62 . As will be understood by artisans in the field, the insulating encapsulation material is selected to have a minimum and preferably no effect on both the electrical and chemical components of structure  60  and the subject tests. A contact layer  82  of metal is deposited on the upper surface of encapsulation layer  80  so as to extend a short distance from the right-hand edge of structure  60  to the left-hand edge where it is exposed to provide easy access as a contact terminal. A layer  84  of n+ doped a-Si is deposited over the upper surface of contact layer  82 . An intrinsic or insulating layer  86  of a-Si is deposited on the upper surface of layer  84  and a layer  88  of p+ doped a-Si is deposited over a central portion of intrinsic or insulating layer  86  to form amorphous silicon photodiode  64  directly overlying MOTFT  62 . An upper contact layer  90  of transparent conductive material, such as ITO or the like, is deposited over p+ a-Si layer  88  and serves as an upper contact for amorphous silicon photodiode  64 . In this specific embodiment, contact layer  90  extends to the left-hand edge of structure  60  where it is exposed to provide easy access as a contact terminal. 
     In this specific example, a substrate for target DNA fragments is formed in overlying relationship to amorphous silicon photodiode  64  as follows. A layer  91  of transparent insulating material is deposited over contact layer  90  across the entire upper surface of structure  60 . A through-hole or via  92  is formed from the upper surface of layer  91  to the upper surface of gate metal  68  and is filled with metal so that an electrical contact with the bottom gate of MOTFT  62  is formed in the upper surface of layer  91 . A layer  93  of transparent conductive material (e.g. ITO or the like) is deposited over the upper surface of layer  91  so as to extend above the area encompassed by p+ a-Si layer  88 . Layer  93  also extends into contact with the metal in via  91  so as to be in electrical contact with the bottom gate of MOTFT  62  and further extends to the outer edge of structure  60  where it is exposed to provide easy access as a contact terminal. A sensing layer  95  of some transparent non-conductive material, such as SiN, tantalum oxide, or the like is deposited over the upper surface of conductive layer  93  and forms a substrate for target DNA fragments  96 . A wall  97  is formed on the upper surface of sensing layer  95  so as encircle the substrate and form a reservoir  94  to contain a liquid  99  containing DNA nucleotides on the upper surface of the substrate. An illumination source is provided above the substrate/sensing layer  95  and reservoir  94 . All of the material between reservoir  94  and photodiode  64  is generally referred to as the ‘bottom’ of reservoir  94  for convenience. 
     Generally, the operation of structure  60  is the same as described above for structure  10 . A labeled nucleotide carried by liquid  99  into enclosure  96  in proximity to the target DNA fragments on substrate/sensing layer  95 , a fluorescence event will or will not occur when the target DNA fragments are illuminated by illumination source  98 , depending on whether the labeled nucleotide is incorporated onto the target DNA strand by the polymerase enzyme. The presence or absence of fluorescence is sensed by photodiode  64  which appears as a signal between contacts  82  and  90  at the left edge of structure  60 . Simultaneously, the incorporation of nucleotides onto the target fragments release hydrogen ions and produce a change in the pH of liquid  99  in enclosure  96 . The change in pH is sensed by a small change in voltage on conductive layer  93  and, consequently, the bottom gate of MOTFT  62 . The small change in voltage on the bottom gate acts similar to a bias so that a larger signal on the top gate is required to activate (i.e. turn ON or turn OFF) MOTFT  62 . Thus, the small signal is essentially amplified which, depending upon the design and construction of MOTFT  62 , can be as much as a factor of 10. 
     Because either structure  10  or structure  60  include an ion sensing MOTFT and a photodiode which both operate on the same nucleotide incorporation occurrence, in the incorporation of a nucleotide that is designated dark (i.e. guanine in the Illumina scheme described above) the incorporation action is verified by a pH change. Of course all other incorporation events sensed by the photodiode are also confirmed or verified by the pH sensor. It is particularly important to note that for the dual detection system to operate correctly, the photodetector and the extended gate of the ion sensing MOTFT must be contiguous (i.e. operating on the same nucleotide incorporation event). 
     In order to further enhance or facilitate the dual detection process, two options in a biochemical bonding or linking process are illustrated in  FIGS. 3 and 4 . Specifically,  FIG. 3  illustrates sequencing primers  100  bound to the surface of a sensing pad, such as substrate/sensing layer  95  of structure  60 . Alternatively,  FIG. 4  illustrates sequencing primers  100  bound to the surface of a bead, such as bead  46  in well  42  of structure  10 . For simplicity of illustration, the following description illustrates sequencing primers  100  bound to a sensing pad  102  (e.g. substrate/sensing layer  95 ). Specifically,  FIGS. 6 through 13  illustrate several steps in a chemical process for improving detection of nucleotide incorporation as a companion with the dual detection structures described above. 
     Beginning with  FIG. 5 , several identical sequencing primers  100  are bound to the surface of sensing pad  102 . Optional photocleavable blocking molecules  104  are attached to the free end of each sequencing primer  100 . Target DNA fragments  106  are then attached to sequencing primers  100 , as illustrated in  FIG. 6 . Optional cleavable blocker  104  is complexed with a sequencing polymerase enzyme  108 , as illustrated in  FIG. 7 . Optional blocking molecules  104  are cleaved using UV light, as illustrated in  FIG. 8  which allows sequencing polymerase enzyme  108  to incorporate matching nucleotides  114  with sequencing primers  100 . Matching nucleotides  114  are blocked with blocking molecules  112  and are labeled with a fluorophore  110 , in the present example green dye, as illustrated in  FIG. 8 . Hydrogen ions  116  are released upon incorporation of nucleotides  114 , as illustrated in  FIG. 9 . Referring to the above description of the dual detection structures, hydrogen ions  116  are detected by MOTFT  62  (in this specific example). Simultaneously, excitation light source (in this example illumination source  98 ) is pulsed to excite the fluorophore for the optical detection event (i.e. photodiode  64  as illustrated in  FIG. 2 ). Excitation light is for example in a range of approximately 495 nm to approximately 520 nm (depending upon the fluorophore). The pulsing of the excitation light source is followed by pulsing of a UV light source which will cleave off the photocleavable blocking molecule  112 , as illustrated in  FIG. 11 . UV light for the cleaving operation is for example approximately 355 nm (near UV). As illustrated by  FIG. 15 , the wavelengths of the excitation light and the UV cleaving light are separated sufficiently to prevent any inadvertent interaction. 
     With cleavable blocking molecule  112  and labeling fluorophores  110  cleaved from sequencing primers  100  and sequencing polymerase enzyme  108  still attached, as illustrated in  FIG. 12 , the process is ready to be repeated with the next nucleotide (starting with the step illustrated in  FIG. 9 ). The process is repeated for each subsequent nucleotide incorporation event. Depending upon the format of the detection process, nucleotides can either be flowed sequentially or some other combination of flows may be utilized. 
     In an alternative embodiment to the preferred method the fluorophore is replaced by an absorbing moiety such as a gold or silver nanoparticle and the like, such that the optical absorption is increased upon the incorporation of a nucleotide. The detected intensity of the illumination by the photodiode is thus reduced by an incorporation event and increased back to the original detection level when the absorbing moiety is cleaved from the incorporated nucleotide. 
     Referring to  FIG. 13  an illumination device  140  is illustrated in which the fluorescent excitation and UV cleavage steps are performed in sequence or simultaneously. In device  140  a pulsed green LED  142  is positioned to direct green light through a dichroic mirror  143  onto sensing pad  102 . A pulsed UV LED  144  is positioned to direct UV light onto a reflecting surface of dichroic mirror  143  which reflects the UV light onto sensing pad  102 . A collimating lens  145  is positioned between dichroic mirror  143  and sensing pad  102  to collimate the green light and UV light as they are directed onto sensing pad  102 . 
     Referring to  FIG. 14 , a device  150  is illustrated in which a UV LED  152  is used with a green phosphor to provide both the fluorescent excitation and UV cleavage steps simultaneously. In device  150  a narrow wavelength green phosphor is deposited on LED  152  in much the same way that white LEDs are currently made. Using a phosphor for the fluorescent excitation has the additional advantage that the phosphor has a narrow emission line (see  FIG. 15 ) that makes it easy to filter out so as to enhance the fluorescent signal to noise ratio. Thus, device  150  simultaneously directs wavelength separated UV light and green fluorescent light through a collimating lens  155  onto sensing pad  102 . 
     Thus, a new and improved detection process and apparatus are disclosed for DNA sequencing. The new and improved detection process and apparatus incorporates both an optical detection process and a process of detecting hydrogen ions that are released when a nucleotide is incorporated onto a target DNA fragment. The detection apparatus, or dual detector, includes a photodetector and the gate of an ion sensing MOTFT that are positioned contiguously (i.e. operating on the same nucleotide incorporation event). The dual detection can, preferably, be performed simultaneously or in the sequential steps of detecting hydrogen emission and then (once a nucleotide incorporation event is confirmed) performing the optical detection or vice versa. From the above, it will be clear that the use of combination detection of DNA incorporation events substantially improves the fidelity of the sequencing process and potentially extends the ‘read’ length of the sequencing process. Additionally, the use of blocking molecules eliminates the issues of homopolymer detection inherent in the well-known ion torrent ion selective detection process. 
     Various changes and modifications to the embodiment herein chosen for purposes of illustration will readily occur to those skilled in the art. To the extent that such modifications and variations do not depart from the spirit of the invention, they are intended to be included within the scope thereof which is assessed only by a fair interpretation of the following claims.