The present invention is further detailed with respect to the following nonlimiting figures. These figures depict only particular processes and apparatuses according to the present invention with variants existing beyond those depicted.
FIG. 1B1 depicts a primer 6 affixed to a support 7, the primer 6 hybridized to a DNA template molecule wherein a polymerizing enzyme 5 recognizes the 3′ end of the template 3, and the amount of recognition element 1 added to the reaction chamber is less than that required to saturate all hybridization sites on the template strands 3;
FIG. 1B2 depicts alternative schematic immobilization of FIG. 1B1 relative to a support wherein DNA template molecule affixed to a support 7, the DNA template molecule hybridized to a primer 6 wherein a polymerizing enzyme 5 recognizes the 3′ end of the template 3, and the amount of recognition element 1 added to the reaction chamber is less than that required to saturate all hybridization sites on the template strands 3;
The present invention relates to a process of identifying individual units in a self-assembling number of elements as they are assembled into a structure. Without limitation, the instant invention is more specifically directed toward sequencing of a deoxyribonucleotide structure into its individual monomer units. Thus, the present invention has utility as a DNA sequencing process and apparatus.
Generally, determining the sequence of a structure containing N different elements requires a system having N sets of chambers. Each of N sets in turn contains N chambers. To initiate the assembly process a different element is placed in each chamber in every set. For example the i-th chamber in every set contains the i-th element. It is appreciated that each element is optionally affixed to a wall or structure or support or to the bottom of a chamber. When the target sequence is unique, i.e. the next element is always different from the previous one, the next step in the process is to put the same element into each chamber of the same set but different elements across sets. That is, all chambers of the i-th set will receive i-th element. Since there are N sets each set containing N chambers, there are N×N possible two element strings. There will be one and only one chamber where a second element will bind to the first element to begin growing the structure. In all other chambers there will be no addition of one element to the other. Thus, by subtraction it is identified what the sequence of the first and second elements are. For example, if the growth of the structure happened in the i-th chamber of the j-th set then the first element in a sequence is the i-th and the second one is the j-th where i and j are integers greater than zero and less than or equal to N.
All sets not containing the chamber wherein a two element structure was formed are discarded such that only one set of N chambers remains. It is appreciated that all chambers are then optionally washed of free unbound elements so that the only remaining structure in the system is the surface bound or solution two element structure. At this point an excess amount of the identified element is added to each chamber of the i-th set so that all chambers contain the same two element structure.
Finding the third and every other element in the sequence is reduced to a simple algorithm. N different elements are added into N chambers to reveal the next element. All chambers are then optionally washed of the elements from all N chambers and the identified element is added to all chambers in excess to grow the structure one more unit. By repeating the steps for every member of the unidentified sequence the element order is easily determined.
In a nonlimiting example, an unidentified DNA template sequence is resolved by the instant inventive process. DNA is comprised of four element types, an adenine, guanine, thiamine, and cytosine. Therefore, the integer N is equal to 4. It is appreciated that DNA is optionally synthesized in vitro in a chamber in the presence of all required molecules illustratively including a DNA polymerase and a helicase. It is further appreciated that with a given sequence of DNA only one of the four types of elements A, T, G or C will be assembled in with the target sequence at each hybridization site being identified. It is known in the art that A hybridizes to T and G hybridizes to C. If the next element in an unknown sequence of DNA is a T only the chamber containing an A recognition element will produce extension, thus, removing the A element from solution. All other chambers will contain free nucleotide. By identifying which wells contain free nucleotide the sequence of the target is deciphered. Thus, according to the inventive process DNA sequencing is illustratively performed using four chambers with the appropriate number of DNA molecule copies in each chamber.
Of primary importance to the inventive process is that each step is conceptually split into many small steps. Each small step consists of supplying one dose of elements wherein the number of DNA template copies is much larger than the number of elements delivered during each small step. Therefore, if a particular element is incorporated into a DNA molecule at the next unoccupied site in the sequence, it is appreciated that the number of free monomers is negligible in solution after the first small step. This process allows simple identification of repeat elements (or copy number of a particular template) in the structure sequence. In a nonlimiting example the total number of DNA copies of template copies in the chamber is ten times larger than the number of elements in each single dose. It is appreciated that the first dose of elements being one-tenth that of the number of DNA will occupy sites on one-tenth of the DNA molecules leaving nine-tenths of the sites on the DNA template molecules free. After ten small additions of elements all sites on the template DNA will be occupied. Between and after each addition one-tenth concentration of element it is appreciated that there will be no free monomer elements in solution because all of the elements will be incorporated into DNA molecules. Upon addition 11 observation of free monomers in the solution occurs which signals completion of the current step and beginning of the next one. As such, a primary advantage of the instant invention over the prior art is a rapid and accurate process for revealing repeat elements or nucleotides in the sequence.
In a nonlimiting example the chamber under consideration contains 100,000 copies of DNA template molecules. One dose of monomer elements contains 10,000 molecules of monomer. Therefore, it will require 10 doses of monomer element to fill the vacancies in all copies of DNA molecules. The eleventh dose will create 10,000 monomer elements available for detection in the solution signaling that the site is not a repeat and, further, signaling the next recognition step.
During sequence identification only one chamber out of four will incorporate elements into the growing DNA structure the other three chambers will demonstrate free monomers in solution, thus, revealing the nature of the monomer which is incorporated into the growing DNA structure. After identification of which monomer element is incorporated at the particular site sufficient copies of that identified monomer element is optionally added to the other three chambers, thus, occupying that site on a growing DNA structure in all four chambers. That completes the final step of the process and determination of the entire sequence of the unknown DNA template molecule is similarly determined.
The elements supplied to the chamber are classified by purpose. The first type of element is a recognizing element. Recognizing elements are elements identifiable as free in solution after being unable to bind in a complementary fashion to the DNA template at the next available site. The second type of element is a building element. Building elements are elements not intended to be used for recognition, however, it is appreciated that they are optionally used as recognizing elements. Building elements are designed to occupy free sites in all growing DNA structures left vacant by the non-complementary recognizing elements in a chamber.
The inventive process is operable for many different types of sequence structures or element containing structures. A recognition element or a building element is illustratively a nucleotide, a ribonucleotide, deoxyribonucleotide, deoxynucleotide, peptide nucleotides, modified nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, amino acids, or modified amino acids. Although chemically similar, a recognition element is mainly involved in an inventive process for nucleotide identification or sequencing while a building element is applied for a collateral determination of copy number of a particular DNA template.
Recognition elements are optionally labeled. A single label or multiple labels are optionally present on each individual recognition element. In a nonlimiting example, during DNA sequencing four different types of recognition elements are employed: A, T, G, or C. Each recognition element optionally contains the same label or different labels that are distinguishable from each other based on characteristics of the combination of label and the remainder of the recognition element. Labels are optionally bound to one or multiple sites on a recognition element such as in a nucleotide. Recognition elements optionally have a label attached at a base, on a sugar moiety, on the alpha phosphate, beta phosphate, gamma phosphate, or any combination thereof. Illustratively, a label for adenine preferably has a fluorophore bound to the gamma phosphate wherein the fluorophore is distinguishable from a fluorophore bound to the gamma phosphate on a different species of recognition element. Thus, in the case of DNA sequence the four recognition element species optionally contain four different fluorophores.
Multiple label types are operable in the instant invention illustratively including cliromophores, fluorescent moieties, enzymes, antigens, dyes, phosphorescent groups, chemiluminescent moieties, scattering or fluorescent nanoparticles, Raman signal generating moieties, fluorescence resonance energy transfer donor or acceptor molecules, precursors thereof, cleavage products thereof, and combinations thereof. In addition, it is appreciated that the label on any recognition element is optionally photo bleachable, photo quenchable, or inactivatable. A recognition element is optionally bound into a single strand of growing DNA in the formation of a structure, and prior to, during, or subsequent to the addition of this recognition element the label is photo bleached such that contamination of the fluorescence of the label does not interfere with subsequent identification steps.
Identifying the presence or absence of free recognition elements in a chamber is dependent on the type of label present on the individual recognition elements. Numerous identifying methods are known in the art illustratively including far field microscopy, near field microscopy, evanescent wave or wave guided illumination, nanostructure enhancement, mass spectroscopy, photon excitation, multi photon excitation, FRET, photo conversion, spectral wavelength discrimination, fluorophore identification, background suppression, electrophoresis, surface plasma in resonance, enzyme reaction, fluorescence lifetime determination, radio frequency modulation, pulsed mutiline excitation, or combinations thereof.
It is appreciated that the structures are optionally complementary to a template structure that guides which element is placed in the next location in the sequence. Illustratively, the template structure is a DNA oligonucleotide sequence. Template DNA sequences are optionally free in solution or bound to a support in a reaction chamber or to the reaction chamber wall itself. Mobilization of the template is accomplished through conventional techniques known in the art illustratively including covalent attachment to a functional group on the solid surface, or by biotin/avidin interaction. In an optional embodiment a short oligonucleotide primer is bound to a support. The oligonucleotide segment is complementary to a small known sequence on the DNA template strand. Hybridization of the DNA template strand with the surface bound oligonucleotide immobilizes the DNA template to the surface of the chamber in reversible fashion. This embodiment has the additional advantage of providing a primer sequence for a polymerization reaction to occur. It is common in the art of DNA sequencing analyses that small segments of known sequence are present at the termination of each unknown strand. The template strand is optionally double stranded DNA, single stranded DNA, single stranded DNA hairpins, RNA, or RNA hairpins.
The inventive process further comprises a polymerization reaction in which one unknown recognition element or building element is added to the growing DNA structure in a complementary fashion. The polymerization reaction is performed by a nucleic acid polymerizing enzyme that is illustratively a DNA polymerase, RNA polymerase, reverse transcriptase, or mixtures thereof. It is further appreciated that accessory proteins or molecules are present to form the replication machinery. In a preferred embodiment the polymerizing enzyme is a thermostable polymerase or thermodegradable polymerase. Use of thermostable polymerases is well known in the art such as Taq polymerase available from Invitrogen Corporation. Thermostable polymerases allow a recognition or building reaction to be initiated or shut down by a change in temperature or other condition in the chamber without destroying activity of the polymerase.
Accuracy of the base pairing in the preferred embodiment of DNA sequencing is provided by the specificity of the enzyme. Error rates for Taq polymerase tend to be false base incorporation of 10−5 or less. Johnson, Annual Reviews of Biochemistry, 1993: 62:685-713; Kunkel, Journal of Biological Chemistry, 1992; 267:18251-18254 (both of which are hereby incorporated by reference.) Specific examples of thermostable polymerases illustratively include those isolated from Thermus aquaticus, Thermus thermophilus, Pyrococcus woesei, Pyrococcus furiosus, Thermococcus litoralis and Thermotoga maritima. Thermodegradable polymerases illustratively include E. coli DNA polymerase, the Klenow fragment of E. coli DNA polymerase, T4 DNA polymerase, T7 DNA polymerase and other examples known in the art. It is recognized in the art that other polymerizing enzymes are similarly suitable illustratively including E. coli, T7, T3, SP6 RNA polymerases and AMV, M-MLV, and HIV reverse transcriptases.
The polymerases are optionally bound to a primer template sequence. When the template sequence is a single-stranded DNA molecule the polymerase is bound at the primed end of the single-stranded nucleic acid at an origin of replication or with double stranded DNA to a nick or gap. Similarly, secondary structures such as in a DNA hairpin or an RNA hairpin allow priming to occur and replication to begin. A binding site for a suitable polymerase is optionally created by an accessory protein or by any primed single-stranded nucleic acid.
In a preferred embodiment the template is bound to a support located within the chamber. Materials suitable for forming a support optionally include glass, glass with surface modifications, silicon, metals, semiconductors, high refractive index dielectrics, crystals, gels and polymers. A support is illustratively a planar or spherical surface. It is appreciated in the inventive process that either a sequencing primer in the case of DNA sequencing, a target nucleic acid molecule, or the nucleic acid polymerizing enzyme are illustratively immobilized on the support. A complementary bonding partner for forming interactions with any of the above molecules or any other of the operational machinery in the inventive process are similarly appreciated to be suitable for immobilizing material onto a surface. Interaction of any of the replication machinery with the surface is optionally nonspecific. Examples of a specific type bonding interaction include a biotin/streptavidin linkage wherein a known primer sequence is optionally labeled with a biotin and the solid support is labeled with a streptavidin. When the biotin primer is added to the chamber a tight bonding interaction between the biotin and streptavidin occurs immobilizing the primer sequence onto the support surface. It is further appreciated that the target DNA sequence is optionally labeled itself so that it is immobilized on the support surface. Additionally, a primer sequence is optionally immobilized by hybridization with a complementary immobilized oligonucleotide. Thus a primary oligonucleotide is immobilized on a surface with a short sequence complementary to the primer oligonucleotide. It is preferred that the primer oligonucleotide is of sufficient additional length that hybridization between the immobilized nucleotide and the primer oligonucleotide allows base pairing between the primer oligonucleotide and the target DNA sequence, thus, binding the target DNA sequence to the support surface. Interaction of any suitable molecule to the support surface is appreciated to be reversible or irreversible. Alternative exemplary methods for immobilizing sequencing primer or target nucleic acid molecule to a support include antibody antigen binding pairs or photoactivated coupling molecules. It is appreciated in the art that numerous other immobilizing methods are similarly suitable in the inventive process.
It is further appreciated that the proteinaceous material of the polymerization enzyme in the case of a DNA polymerase is optionally immobilized on the surface either reversibly or irreversibly. For example, RNA polymerase was successfully immobilized on activated surface without loss of catalytic activity. Yin et al., Science, 1995; 270: 1653-57, which is hereby incorporated by reference. Alternatively, an antibody antigen pair is utilized to bind a polymerase enzyme to a support surface whereby the support surface is coated with an antibody that recognizes an epitope on the protein antigen. When the antigen is introduced into the reaction chamber it is reversibly bound to the antibody and immobilized on the support surface. A lack of interference with catalytic activity in such a method has been reported for HIV reverse transcriptase. Lennerstrand, Analytical Biochemistry, 1996; 235:141-152, which is hereby incorporated by reference. Additionally, DNA polymerase immobilization has been reported as a functional immobilization method in Korlach et al., U.S. Pat. No. 7,033,764 B2; incorporated herein by reference. Finally, any protein component can be biotinylated such that a biotin streptavidin interaction is optionally created between the support surface and the target immobilized antigen.
In a preferred embodiment both the target and the polymerase remain free in solution. Referring to
In the preferred embodiment depicted in
Each chamber is optionally in fluidic connection with a detector such that by washing each of the chambers free recognition elements are transported to the detector area and are readily detected. In a preferred embodiment each of the recognition elements is differentially labeled such that it can be easily distinguished from other recognition elements. Thus, a single detector is employed whereby the individual unincorporated element species are readily identified, thus, determining the sequence at the first hybridization site in the template molecule.
As depicted in
Alternatively, numerous collection chambers are optionally employed. In a nonlimiting example each reaction chamber serves the additional roles of repeat detection chamber and sequence building chamber. Following addition of recognition elements to the recognition chambers, a first electric potential is applied to move all free recognition elements past a detector to identify the next element in sequence. This removes all unbound recognition elements from the reaction chambers. A portion or all of the reaction chambers are then used as repeat detection chambers whereby additional recognition elements are optionally added to determine the repeat number if any. A second electric potential is then applied to remove all unbound elements from the recognition chambers to a second set of collection areas. The first electric potential is then applied with reverse polarity to move all the unhybridized recognition elements back into their original recognition chambers negating the need to add more recognition elements to the N−1 chambers that did not demonstrate hybridization, thus, saving reagent and expense.
In an alternative embodiment a sampling of each of the reaction chambers or the repeat detecting chamber is obtained and injected into a mass spectrometer to recognize the presence of free elements. This embodiment has the advantage of using native, non-labeled elements whereby greater efficiency and accuracy of the polymerase is achieved. Alternatively, it is appreciated that multiple detector types are optionally employed. In a nonlimiting example, the recognition elements are fluorescently labeled. Detection of the species of hybridizing recognition element is, thus, detected by a fluorometer. After washing all unbound recognition elements from the reaction chambers, repeat detection is accomplished by addition of unlabeled recognition elements or building elements. Free elements are optionally detected by a mass spectrometer. This has the advantage of allowing washing of the chamber and use of N chambers for recognition, repeat detection, and building. Also, use of unlabeled elements in the repeat detection phase allows N replicates of repeat detection without contamination of the next round of sequence recognition.
In a preferred embodiment the template is bound to a support. Washing of the chambers removes only unbound recognition element. In an alternative embodiment the fluidic connection between each reaction chamber in the detector is such that the large template molecule remains in the chamber while the small recognition elements are readily transported through a barrier such as a size exclusion membrane or an electrophoretic gel. As such, each chamber is washed free of unbound recognition elements.
Once the complementary species of the recognition element is identified, this recognition element species is optionally stepwise added to each of the four chambers. In a nonlimiting example one-tenth concentration of recognition element was initially added to each of the four chambers for identification purposes. Stepwise addition of one-tenth concentration recognition elements allows for gradual saturation of all hybridization sites on each of the template strands. Should the stepwise additions exceed a value of ten it is understood that there is a repeat. For example if 20 stepwise additions are required before saturation of all hybridization sites on the template molecule occurs, it is appreciated that there is a single repeat on the template strand.
After identification of the particular recognition element species bound to the template molecule and determination of whether or not a repeat of that particular recognition element species occurs, building elements are added to each of the chambers at a known concentration to fully saturate all structures. At this point all unbound recognition element and building element species are optionally washed from each chamber and the reaction cycle begins again so as to determine the next recognition element species in each of the template strands. Thus, by repeating the sequence of steps the sequencing primer is extended and the entire sequence of the target is determined.
It is appreciated that the solution be of suitable extension medium so as to permit diffusion, incorporation, and washing out of each of the reaction chambers. In a nonlimiting example suitable extension media a contains 50 mM Tris-HCl pH 8.0, 25 mM magnesium chloride, 65 mM sodium chloride, 3 mM DTT, and elements at appropriate concentration to permit identification of the sequence. It is appreciated that other extension medium are similarly suitable and optimized for the particular polymerase or template being utilized.
In an alternative embodiment as in the present nonlimiting illustration, a fifth chamber is present termed a repeat detecting chamber wherein, following identification of the recognition element species, recognition element species is added to the repeat detecting chamber to determine whether or not a repeat exists and the number of repeats in sequence. Following identification of both the recognition element species in the original reaction chambers as well as the number of repeats in the repeat detecting chamber, a suitable concentration of building elements is added to all chambers to fully saturate all sites at that portion in the growing structure. It is appreciated that a washing out procedure is optionally employed between each subsequent sequence whereby the unbound elements in all reaction chambers are removed.
In an alternative embodiment the repeat detecting chamber is in fluidic communication with each of the four reaction chambers. All the reaction solution from each of the four reaction chambers is transferred to the repeat detecting chamber. It is in the repeat detecting chamber that stepwise addition of the identified species of recognition element is added to determine whether or not a repeat exists. Once the presence of a repeat is determined, or shown not to exist, the repeat detecting chamber is optionally washed free of all unbound recognition elements and the fully hybridized growing DNA molecule is subsequently transferred back to each of the four reaction chambers for the next round of element recognition.
In an alternative embodiment there is no washing out of the elements which are left in solution as excessive free elements after each of the previous steps. However, it is appreciated that the ratio between recognition elements and template is such that there is little to no observable contamination as the procedure moves through several rounds of recognition. For example, in a situation with five chambers, four recognition chambers and a repeat detecting chamber, four contain copies of free DNA molecules to be sequenced. Each chamber is initially supplied only with a small dose of one species of recognition element. This small dose is illustratively one-tenth concentration of target DNA molecules to be sequenced. After identification of the next hybridizing recognition element that element is added to the fifth chamber only using small doses to determine if there is a repeat. After the correct dose of that element is determined, the appropriate concentration of building element is added to the first four chambers and the next round of recognition begins.
In yet another alternative embodiment a sixth chamber is present termed a sequence construction chamber. Four recognition chambers contain copies of free DNA molecules to be sequenced, and each chamber is supplied with only a single species of recognition element. After the next hybridizing element is identified as described in the previous procedures, that element is added to the fifth repeat detecting chamber to determine if there is a repeat. Subsequently, the solution from all five chambers is then moved to a sixth chamber where the appropriate number of building elements for all six chambers is added to the resulting solution so as to fully saturate all free sites in the growing structure that are complementary to the current hybridization site on the template. Following saturation of all sites the solution from the sequence construction chamber transferred back to each of the four recognition chambers and the repeat detecting chamber. All chambers now contain a DNA template molecule hybridized to a growing structure of equal length and a new round of recognition element species identification occurs.
In an alternative embodiment, after identification of the next complementary recognition element each of the four recognition chambers is emptied and washed such that the solution from each of the four chambers is fully discarded and not sent to the fifth or sixth chamber. The correct amount of the determined element is added to the fifth chamber, which is a large chamber, so as to fully saturate all hybridization sites on the template DNA molecule in this chamber. Small volumes of the fifth repeat detection chamber are subsequently added back to each of the recognition chambers for a new round of recognition species identification. Optionally, a washing out procedure occurs during transfer of solution from each of the recognition chambers to a fifth repeat detecting chamber.
In an alternative process employing six chambers, five small chambers and one large sequence construction chamber, five small volumes of target DNA molecules in solution, or immobilized on a support in suspension, are transferred from the sequence construction chamber to each of the five other chambers. The next element in sequence is determined as described above. The fifth chamber is then used to determine the number of possible repeats. After the recognition element species is identified and the number of repeats is determined, the solution from all five chambers is transferred back to the sixth chamber where a correct amount of the determined element is added and the whole procedure is then repeated. It is appreciated that in this embodiment simultaneous sequencing and amplification of target DNA occurs. For example, in the situation where one large sixth sequence construction chamber is utilized small samples are withdrawn and divided amongst the four recognition chambers and the fifth repeat detecting chamber. The next element in series is identified and the presence of repeats is determined. The proper dose of building elements is then added to the sequence construction chamber to fully saturate all sites on the growing DNA structure.
It is appreciated that numerous other embodiments of the instant invention exist with greater or fewer chamber numbers, types, sizes, interconnections, or pathways and are also the subject of the instant invention.
An embodiment of the instant invention includes an apparatus. This apparatus optionally employs numerous reactor types illustratively including a batch reactor, a plug flow reactor, or a drop reactor. An apparatus for self-assembly of a number of elements comprises a reaction area that contains a suitable number of chambers relative to the number of different species of elements in the growing structure; a preparation area in fluidic connection with the reaction area whereby reagents and solutions are prepared to be delivered to the reaction area in stepwise or simultaneous fashion; and a detection area in fluidic, physical, or optical connection with the reaction area.
The detection area employs any suitable detector for detection of the type of label on each of the individual recognition elements. For example, if each of the recognition elements is labeled with a particular fluorophore a fluorescent detector is employed so as to identify which chambers contain free recognition elements. In the case where either unlabeled recognition elements are employed or nonoptically resolvable recognition elements are employed each of the reaction chambers is optionally connected to a mass spectrometer whereby the presence of free recognition elements is readily determined.
In the inventive apparatus the reaction area has N recognition chambers, each chamber having a plurality of microdispensers. The number of microdispensers is related to the number of possible recognition element species. For example, if there are four recognition element species each chamber in the reaction area has four microdispensers to allow distribution of the various species of recognition element. In an alternative embodiment there are eight microdispensers aimed at each of the reaction chambers such that any of the four recognition elements are optionally distributed to each reaction chamber as well as any building elements without fear of contamination between the elements. Thus, each microdispenser is filled with one type of element so that each type of element is available to be distributed into each chamber in the reaction area. In the case of five small chambers the fifth chamber similarly has four or eight microdispensers for delivery of elements to that chamber. It is appreciated that the number of microdispensers is optionally related to the number of the elements in the growing structure. In the case of ten separate element species as many as ten or twenty microdispensers for each chamber are employed. Alternatively, a single or fewer than N microdispensers is employed with a washing out step of each of the microdispensers between delivery of different recognition or building elements.
It is appreciated that the reaction area contains no moving parts. Fluidic connection between each of the chambers is optionally powered by differential electric potential so as to move free recognition or building elements between the chambers. Further, DNA template and growing structure may similarly be transferred between chambers.
A standard reaction chamber protocol is outlined in
Fluorescein-12 labeled A is added to the repeat detection chamber along with DNA polymerase, MgSO4, and reaction solution by a microdispenser in 1/10 mol/mol amounts in sequential fashion and the reaction is allowed to proceed for 5 sec followed by application of an electric potential to determine if free nucleotide is present in solution. It is appreciated that other relative amounts of nucleotide and template are similarly suitable in all chambers. Application of an electric potential moves free nucleotide to a detector area where the presence of free nucleotide is determined as above. The process in the repeat detection chamber is repeated until free nucleotide recognized by the detector. Twenty additions are required for the instant exemplary template strand indicating that there is an AA repeat sequence.
2×mol/mol concentration of unlabeled A nucleotide (building element) is added to each of the reaction chambers and the sequence building chamber and the polymerization is allowed to proceed for 2 min followed by application of an electric potential to wash out any remaining free recognition or building element from all chambers.
The process is repeated for 350 cycles to fully assemble and identify the sequence of all nucleotide elements in the DNA sequence.
Referring to
Patent documents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. These documents and publications are incorporated herein by reference to the same extent as if each individual document or publication was specifically and individually incorporated herein by reference.
The foregoing description is illustrative of particular embodiments of the invention, but is not meant to be a limitation upon the practice thereof. The following claims, including all equivalents thereof, are intended to define the scope of the invention.
This application claims the benefit of U.S. Provisional Patent Applications 60/836,103 filed on Aug. 7, 2006; and 60/905,357 filed on Mar. 7, 2007; the entire disclosures of which are hereby incorporated herein by reference.
Number | Date | Country | |
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60836103 | Aug 2006 | US | |
60905357 | Mar 2007 | US |