TOPACIO-HALL DENISE SANCHEZ (US)
TOZER EILEEN COLLINS (US)
US20180142285A1 | 2018-05-24 | |||
US20120070838A1 | 2012-03-22 |
CLAIMS What is claimed is: 1. A method for monitoring a nucleotide incorporation reaction, the method comprising: forming a reaction mixture comprising: a template oligonucleotide sequence comprising a primer binding site, one or more nucleotide residues, and a nucleotide residue at a 5’ end of the one or more nucleotide residues that is different from the one or more nucleotide residues; a primer sequence configured to hybridize to the primer binding site; a first nucleotide set comprising one or more nucleotides that are complementary to the one or more nucleotide residues, wherein all or a portion of the one or more complementary nucleotides comprise a first member of a fluorescence probe pair; a second nucleotide that is complementary to the nucleotide residue at the 5’ end of the one or more nucleotide residues and that comprises a second member of the fluorescence probe pair; and a polymerase; and detecting a presence or absence of a fluorescence resonance energy transfer (FRET) signal, wherein a change in the FRET signal indicates that the polymerase has incorporated nucleotides of the first nucleotide set and the second nucleotide to extend the primer sequence through the one or more nucleotide residues and the nucleotide residue at the 5’end of the one or more nucleotide residues. 2. The method of claim 1, wherein the one or more nucleotide residues comprise a single nucleotide residue. 3. The method of claim 1, wherein the one or more nucleotide residues comprise a homopolymer sequence. 4. The method of claim 1, wherein the one or more nucleotide residues comprise a short tandem repeat sequence. 5. The method of claim 4, wherein the short tandem repeat sequence comprises two or more dinucleotide repeats. 6. The method of claim 4, wherein the short tandem repeat sequence comprises two or more trinucleotide repeats. 7. The method of claim 4, wherein a repeat pattern for the short tandem repeat sequence is from 2 to 16 nucleotides in length, and the short tandem repeat comprises from 2 to 10 repeats of the repeat pattern. 8. The method of claim 1, wherein the one or more nucleotide residues comprise a random sequence of nucleotide residues ranging from 2 to 50 nucleotide residues in length. 9. The method of any one of claims 1 to 8, wherein the FRET signal is detected at a fixed point in time following the formation of the reaction mixture. 10. The method of any one of claims 1 to 8, wherein the FRET signal is detected as a function of time following addition of the polymerase to the reaction mixture. 11. The method of any one of claims 1 to 10, wherein all of the one or more complementary nucleotides of the first nucleotide set comprise the first member of the fluorescence probe pair. 12. The method of any one of claims 1 to 10, wherein at least one complementary nucleotide of the first nucleotide set comprises the first member of the fluorescence probe pair, and at least one complementary nucleotide of the first nucleotide set does not comprise the first member of the fluorescence probe pair. 13. The method of any one of claims 1 to 10, wherein at least one type of complementary nucleotide included in the first nucleotide set comprises a mixture of nucleotides that are labeled with the first member of the fluorescence probe pair and nucleotides that are not labeled with the first member of the fluorescence probe pair. 14. The method of any one of claims 1 to 13, wherein the fluorescence probe pair comprises a fluorescence donor-acceptor pair, and an increase in FRET signal indicates that the polymerase has incorporated the first set of nucleotides and the second nucleotide to extend the primer sequence through the one or more nucleotide residues and the nucleotide residue at the 5’end of the one or more nucleotide residues. 15. The method of claim 14, wherein the first member of the fluorescence donor-acceptor pair is a fluorescence donor, and the second member of the fluorescence donor-acceptor pair is a fluorescence acceptor. 16. The method of claim 14 or claim 15, wherein the fluorescence donor-acceptor pair is FITC - Rhodamine, Alexa488 - Cy3, Cy3 - Cy5, Atto550–Atto647N, Alexa546 - Alexa647, Pacific Blue- Atto532, or Atto532 - Atto633. 17. The method of any one of claims 1 to 13, wherein the fluorescence probe pair comprises a fluorescence donor-quencher pair, and a decrease in FRET signal indicates that the polymerase has incorporated the first set of nucleotides and the second nucleotide to extend the primer sequence through the one or more nucleotide residues and the nucleotide residue at the 5’end of the one or more nucleotide residues. 18. The method of claim 17, wherein the first member of the fluorescence donor-quencher pair is a fluorescence donor, and the second member of the fluorescence donor-quencher pair is a fluorescence quencher. 19. The method of claim 17 or claim 18, wherein the fluorescence donor-quencher pair is Quasar670 – BHQ2, CalRed – BHQ2, Quasar570 – BHQ2, TET – BHQ2, or TAMRA – BHQ2. 20. A method for monitoring a nucleotide incorporation reaction, the method comprising: forming a reaction mixture comprising: a template oligonucleotide sequence comprising a primer binding site, one or more nucleotide residues, a nucleotide residue at a 5’ end of the one or more nucleotide residues that is different from the one or more nucleotide residues, and a first fluorescence probe; a primer sequence configured to hybridize to the primer binding site; a first nucleotide set comprising one or more nucleotides that are complementary to the one or more nucleotide residues, wherein all or a portion of the one or more complementary nucleotides comprise a second fluorescence probe; a second nucleotide that is complementary to the nucleotide residue at the 5’ end of the one or more nucleotide residues and that comprises a third fluorescence probe; and a polymerase; and detecting a presence or absence of a fluorescence resonance energy transfer (FRET) signal, wherein a change in the FRET signal indicates that the polymerase has incorporated nucleotides of the first nucleotide set and the second nucleotide to extend the primer sequence through the one or more nucleotide residues and the nucleotide residue at the 5’end of the one or more nucleotide residues. 21. The method of claim 20, wherein the one or more nucleotide residues comprise a single nucleotide residue. 22. The method of claim 20, wherein the one or more nucleotide residues comprise a homopolymer sequence. 23. The method of claim 20, wherein the one or more nucleotide residues comprise a short tandem repeat sequence. 24. The method of claim 23, wherein the short tandem repeat sequence comprises two or more dinucleotide repeats. 25. The method of claim 23, wherein the short tandem repeat sequence comprises two or more trinucleotide repeats. 26. The method of claim 23, wherein a repeat pattern for the short tandem repeat sequence is from 2 to 16 nucleotides in length, and the short tandem repeat comprises from 2 to 10 repeats of the repeat pattern. 27. The method of claim 20, wherein the one or more nucleotide residues comprise a random sequence of nucleotide residues ranging from 2 to 50 nucleotide residues in length. 28. The method of any one of claims 20 to 27, wherein the FRET signal is detected at a fixed point in time following the formation of the reaction mixture. 29. The method of any one of claims 20 to 27, wherein the FRET signal is detected as a function of time following addition of the polymerase to the reaction mixture. 30. The method of any one of claims 20 to 29, wherein all of the one or more complementary nucleotides of the first nucleotide set comprise the second fluorescence probe. 31. The method of any one of claims 20 to 29, wherein at least one complementary nucleotide of the first nucleotide set comprises the second fluorescence probe, and at least one complementary nucleotide of the first nucleotide set does not comprise the second fluorescence probe. 32. The method of any one of claims 20 to 29, wherein at least one type of complementary nucleotide included in the first nucleotide set comprises a mixture of nucleotides that are labeled with the second fluorescence probe and nucleotides that are not labeled with the second fluorescence probe. 33. The method of any one of claims 20 to 32, wherein the first fluorescence probe and the second fluorescence probe form a first fluorescence donor – acceptor pair. 34. The method of any one of claims 20 to 33, wherein the second fluorescence probe and the third fluorescence probe form a second fluorescence donor – acceptor pair. 35. The method of any one of claims 20 to 34, wherein the first fluorescence probe is a fluorescence donor for the second fluorescence probe, and the second fluorescence probe is a fluorescence donor for the third fluorescence probe. 36. The method of any one of claims 20 to 35, wherein the first fluorescence probe comprises Pacific Blue. 37. The method of any one of claims 20 to 36, wherein the second fluorescence probe comprises Atto532. 38. The method of any one of claims 20 to 37, wherein the third fluorescence probe comprises Atto633. 39. The method of any one of claim 20 to 32, wherein at least one of the second or third fluorescence probes is a fluorescence quencher. 40. The method of any one of claims 20 to 39, further comprising exciting the first fluorescence probe with light of an appropriate wavelength and measuring a fluorescence emission intensity for the first fluorescence probe as a function of time. 41. The method of claim 40, wherein a decrease in the fluorescence emission intensity for the first fluorescence probe indicates incorporation of a nucleotide of the first nucleotide set. 42. The method of claim 40 or claim 41, further comprising measuring a FRET-based fluorescence emission intensity for the second fluorescence probe as a function of time. 43. The method of claim 42, wherein a transient increase in the FRET-based fluorescence emission intensity for the second fluorescence probe indicates initial incorporation of nucleotides of the first nucleotide set followed by incorporation of the second nucleotide. 44. The method of any one of claims 40 to 43, further comprising measuring a FRET-based fluorescence emission intensity for the third fluorescence probe as a function of time. 45. The method of claim 44, wherein an increase in the FRET-based fluorescence emission for the third fluorescence probe indicates incorporation of the second nucleotide. 46. The method of any one of claims 1 to 45, wherein the one or more nucleotide residues comprise one, two, three, four, five, six, seven, eight, nine, or ten nucleotide residues. 47. The method of any one of claims 1 to 46, wherein the polymerase comprises a DNA polymerase, an RNA polymerase, or a reverse transcriptase. 48. The method of any one of claims 1 to 47, wherein the reaction mixture is free of nucleotides that do not comprise a fluorescence probe. 49. The method of any one of claims 1 to 47, wherein the reaction mixture further comprises at least one non-labeled nucleotide. 50. The method of any one of claims 1 to 49, further comprising repeating the method using a plurality of template oligonucleotide sequences for which the one or more nucleotide residues comprise a different number of nucleotide residues of the same type for each template oligonucleotide sequence of the plurality, and comparing FRET signals detected for the plurality of template oligonucleotide sequences to determine an effective maximum homopolymer length for which the polymerase is capable of efficiently incorporating labeled complementary nucleotides. 51. The method of any one of claims 1 to 50, further comprising repeating the method using a plurality of template oligonucleotide sequences for which the one or more nucleotide residues comprise a different number of nucleotide residues for each template oligonucleotide sequence of the plurality, and comparing FRET signals detected for the plurality of template oligonucleotide sequences to determine an ability of the polymerase to sustain continuous primer extension reactions using labeled nucleotides. 52. The method of any one of claims 1 to 51, further comprising determining a rate of change of FRET signal following the formation of the reaction mixture. 53. The method of claim 52, wherein the method is used to screen a library of mutant polymerases and rank order the mutant polymerases based on the determined rate of change of the FRET signal. 54. The method of claim 52, further comprising calculating an average nucleotide incorporation rate for the polymerase from the determined rate of change of the FRET signal and a known length of a sequence segment comprising the one or more nucleotide residues. 55. The method of claim 54, wherein the method is used to screen a library of mutant polymerases and rank order the mutant polymerases according to calculated average nucleotide incorporation rates. 56. The method of claim 53 or claim 55, further comprising selecting a mutant polymerase from the library of mutant polymerases based on its rank order. 57. The method of claim 56, wherein the selected mutant polymerase is more efficient than a corresponding wild-type polymerase at incorporating nucleotides into a growing nucleic acid strand. 58. The method of claim 57, wherein the selected mutant polymerase is more efficient than the corresponding wild-type polymerase at incorporating labeled nucleotides into a growing nucleic acid strand. 59. The method of any one of claims 56 to 58, wherein the selected mutant polymerase is less likely than a corresponding wild-type polymerase to misincorporate nucleotides during nucleic acid sequencing. 60. The method of claim 59, wherein the selected mutant polymerase is less likely than the corresponding wild-type polymerase to misincorporate labeled nucleotides during nucleic acid sequencing. 61. A method for screening a library of mutant polymerases, the method comprising: forming a reaction mixture comprising: a template oligonucleotide sequence comprising a primer binding site, one or more nucleotide residues, and a nucleotide residue at a 5’ end of the one or more nucleotide residues that is different from the one or more nucleotide residues; a primer sequence configured to hybridize to the primer binding site; a first nucleotide set comprising one or more nucleotides that are complementary to the one or more nucleotide residues, wherein all or a portion of the one or more complementary nucleotides comprise a first member of a fluorescence probe pair; a second nucleotide that is complementary to the nucleotide residue at the 5’ end of the one or more nucleotide residues and that comprises a second member of the fluorescence probe pair; and a mutant polymerase from the library of mutant polymerases; and detecting a presence or absence of a fluorescence resonance energy transfer (FRET) signal, wherein a change in the FRET signal indicates that the mutant polymerase has incorporated nucleotides of the first nucleotide set and the second nucleotide to extend the primer sequence through the one or more nucleotide residues and the nucleotide residue at the 5’end of the one or more nucleotide residues. 62. A method for screening a library of mutant polymerases, the method comprising: forming a reaction mixture comprising: a template oligonucleotide sequence comprising a primer binding site, one or more nucleotide residues, a nucleotide residue at a 5’ end of the one or more nucleotide residues that is different from the one or more nucleotide residues, and a first fluorescence probe; a primer sequence configured to hybridize to the primer binding site; a first nucleotide set comprising one or more nucleotides that are complementary to the one or more nucleotide residues, wherein all or a portion of the one or more complementary nucleotides comprise a second fluorescence probe; a second nucleotide that is complementary to the nucleotide residue at the 5’ end of the one or more nucleotide residues and that comprises a third fluorescence probe; and a mutant polymerase from the library of mutant polymerases; and detecting a presence or absence of a fluorescence resonance energy transfer (FRET) signal, wherein a change in the FRET signal indicates that the mutant polymerase has incorporated nucleotides of the first nucleotide set and the second nucleotide to extend the primer sequence through the one or more nucleotide residues and the nucleotide residue at the 5’end of the one or more nucleotide residues. 63. The method of claim 61 or claim 62, wherein the one or more nucleotide residues comprise a single nucleotide residue. 64. The method of claim 61 or claim 62, wherein the one or more nucleotide residues comprise a homopolymer sequence. 65. The method of claim 61 or claim 62, wherein the one or more nucleotide residues comprise a short tandem repeat sequence. 66. The method of claim 65, wherein the short tandem repeat sequence comprises two or more dinucleotide repeats. 67. The method of claim 65, wherein the short tandem repeat sequence comprises two or more trinucleotide repeats. 68. The method of claim 65, wherein a repeat pattern for the short tandem repeat sequence is from 2 to 16 nucleotides in length, and the short tandem repeat comprises from 2 to 10 repeats of the repeat pattern. 69. The method of claim 61 or claim 62, wherein the one or more nucleotide residues comprise a random sequence of nucleotide residues ranging from 2 to 50 nucleotide residues in length. 70. The method of any one of claims 61 to 69, wherein the FRET signal is detected at a fixed point in time following the formation of the reaction mixture. 71. The method of any one of claims 61 to 70, wherein the FRET signal is detected as a function of time following addition of the mutant polymerase to the reaction mixture. 72. The method of any one of claims 61 to 71, further comprising repeating the method for at least one additional mutant polymerase. 73. The method of any one of claims 61 to 72, further comprising repeating the method using a plurality of template oligonucleotide sequences for which the one or more nucleotide residues comprise a different number of nucleotide residues of the same type for each template oligonucleotide sequence of the plurality, and comparing FRET signals detected for the plurality of template oligonucleotide sequences to determine an effective maximum homopolymer length for which the mutant polymerase is capable of efficiently incorporating complementary nucleotides. 74. The method of any one of claims 61 to 73, further comprising repeating the method using a plurality of template oligonucleotide sequences for which the one or more nucleotide residues comprise a different number of nucleotide residues for each template oligonucleotide sequence of the plurality, and comparing FRET signals detected for the plurality of template oligonucleotide sequences to determine an ability of the mutant polymerase to sustain continuous primer extension reactions using labeled nucleotides. 75. The method of any one of claims 61 to 74, further comprising determining a rate of change of the FRET signal following the formation of the reaction mixture. 76. The method of claim 75, further comprising rank ordering the mutant polymerases of the library of mutant polymerases according to the rate of change of the FRET signal detected following addition of the mutant polymerase. 77. The method of claim 75, further comprising calculating an average nucleotide incorporation rate for the mutant polymerase from the determined rate of change of the FRET signal and a known length of a sequence segment comprising the one or more nucleotide residues. 78. The method of claim 77, further comprising rank ordering the mutant polymerases of the library of mutant polymerases according to calculated average nucleotide incorporation rates. 79. The method of claim 76 or claim 78, further comprising selecting a mutant polymerase based on its rank order. 80. The method of claim 79, wherein the selected mutant polymerase is more efficient than a corresponding wild-type polymerase at incorporating nucleotides into a growing nucleic acid strand. 81. The method of claim 80, wherein the selected mutant polymerase is more efficient than the corresponding wild-type polymerase at incorporating labeled nucleotides into a growing nucleic acid strand. 82. The method of any one of claims 79 to 81, wherein the selected mutant polymerase is less likely than a corresponding wild-type polymerase to misincorporate nucleotides during nucleic acid sequencing. 83. The method of claim 82, wherein the selected mutant polymerase is less likely than the corresponding wild-type polymerase to misincorporate labeled nucleotides during nucleic acid sequencing. 84. The method of any one of claims 61 to 83, wherein the method is performed in a 6-well, 12-well, 24-well, 48-well, 96-well, 384-well, or 1536-well microplate format. 85. The method of any one of claims 61 to 84, wherein mutant polymerases are screened at a throughput of at least 100 mutant polymerases tested per hour. 86. The method of any one of claims 61 to 85, wherein the mutant polymerases are screened at a throughput of at least 1,000 mutant polymerases tested per hour. 87. The method of any one of claims 61 to 86, wherein the one or more nucleotide residues comprises one, two, three, four, five, six, seven, eight, nine, or ten nucleotide residues. 88. The method of any one of claims 61 to 87, wherein the mutant polymerase comprises a DNA polymerase, an RNA polymerase, or a reverse transcriptase. 89. The method of any one of claims 61, or 63 to 88, wherein the first member of the fluorescence probe pair is a fluorescence donor, and the second member of the fluorescence probe pair is a fluorescence acceptor. 90. The method of any one of claims 62 to 88, wherein the first fluorescence probe is a fluorescence donor for the second fluorescence probe, and the second fluorescence probe is a fluorescence donor for the third fluorescence probe. 91. The method of claim 90, wherein the first fluorescence probe comprises Pacific Blue, the second fluorescence probe comprises Atto532, and the third fluorescence probe comprises Atto633. |
[0058] Use of the FRET technique allows one to quantitatively detect molecular interactions over distances of tens of angstroms. The center-to-center distance between adjacent nucleotide pairs in double-stranded DNA is 3.4 nm (34 angstroms). [0059] Design of FRET-base polymerase assays: A typical DNA polymerase reaction involves an oligonucleotide substrate (usually a hybrid of a bound primer and a template oligonucleotide, e.g., a DNA molecule), the DNA polymerase, and incoming deoxyribonucleoside triphosphates (dNTPs). In the course of the polymerase catalyzed reaction, the enzyme incorporates the incoming dNTPs into the extended primer molecule in a template-dependent manner. There are several different approaches by which the FRET technique can be utilized to detect nucleotide incorporation. For example, in some instances, either the primer sequence or the template DNA molecule can be labeled with one member of the fluorescence probe pair (or FRET donor – acceptor pair) – either the fluorescence donor or the fluorescence acceptor – and the incoming dNTPs can be labeled with the other member. Any nucleotide incorporation event catalyzed by the polymerase is then detected by an increase in FRET efficiency between the two fluorophores (e.g., as a decrease in donor fluorescence or an increase in acceptor fluorescence). Alternatively, in some instances, both components (or members) of the FRET probe pair can be attached to the DNA template/primer complex, and the incoming nucleotides can be unlabeled. In this case, the successful incorporation of the incoming nucleotides causes conformational changes of the primer/template complexes that result in measurable perturbation of the FRET efficiency. In yet another possible configuration, one of the FRET partner dyes can be attached to the polymerase, while the other can be attached to the template/primer hybrid or to the incoming dNTP. In this case, successful incorporation may cause a translocation of the labeled polymerase or bring the incoming labeled nucleotide in close proximity to the polymerase-bound dye, resulting in a measurable change in FRET efficiency. [0060] Disclosed herein are homogeneous polymerase assays designed to evaluate the ability of various DNA polymerases (or other polymerases, e.g., RNA polymerases, reverse transcriptases, and the like) to sequentially incorporate labeled dNTPs into the growing primer molecule. Initial experiments were performed using a primer molecule labeled with a single donor and dNTPs labeled with a suitable acceptor. FRET theory predicts an increase of FRET efficiency with an increasing number of fluorescence acceptor moieties located within the effective FRET distance (e.g., approximately the Förster distance) of the fluorescence donor. In practice, however, it was difficult to differentiate between multiple sequential incorporation events (e.g., in a homopolymer sequence or in a long oligonucleotide region) using this initial assay format. [0061] An alternative assay format, where both the fluorescence donor and the fluorescence acceptor are located on the incoming nucleotides (the other components of the assay system – the template DNA and the polymerase enzyme – remain unlabeled), provided improved ability to detect multiple sequential nucleotide incorporation events used dye-labeled dNTPs. The template oligonucleotide sequences (template DNA strands) used in the assay are designed to enable the incorporation of multiple fluorescence donor-labeled dNTPs first (e.g., in a homopolymer sequence), followed by eventual incorporation of a fluorescence acceptor-labeled dNTP molecule, whereupon a detectable FRET signal between the donor(s) and acceptor is registered. In a reaction mixture comprising a plurality of primed template oligonucleotides undergoing asynchronous polymerase binding and polymerase-catalyzed nucleotide incorporation, the eventual incorporation of the fluorescence acceptor-labeled dNTP molecule into the plurality of primer extension strands leads to an increase in acceptor fluorescence intensity as a function of time, where the rate of increase is proportional to an average incorporation rate for the labeled nucleotides (under conditions that polymerase binding to the primed template molecule is not rate-limiting). [0062] In one non-limiting example, the primer/template hybrid sequences listed in Table 2 were used to evaluate the ability of different polymerases to sequentially incorporate one, two, three, or four dye-labeled dGTPs. Table 2. Non-limiting examples of paired primer / oligonucleotide template sequences. Sequence # C residues Primer / oligo template pair Example No. in template 1 1 …..AGGCT 3’ …..TCCGACA…. 5’ 2 2 …..AGGCT 3’ …..TCCGACCA…. 5’ 3 3 …..AGGCT 3’ …..TCCGACCCA….5’ 4 4 …..AGGCT 3’ …..TCCGACCCCA.. 5’ (SEQ ID NO: 1) [0063] FIG. 2A illustrates the assay format using the third primer / template oligonucleotide sequence pair listed in Table 2. As illustrated in FIG. 2A, the incoming labeled dNTPs used in this example were G* (a dGTP labeled with a fluorescence donor, e.g., Atto532) and U @ (a dUTP labeled with a fluorescence acceptor, e.g., Atto633). The nucleotide incorporation reaction begins following the formation of a reaction mixture (FIG. 2A, upper) comprising the template oligonucleotide sequence, primer sequence, the labeled complementary dNTPs, and the polymerase. Initially, only the labeled-dGTP derivative is incorporated by the polymerase (FIG. 2A, middle). These initial incorporation events remain undetectable by FRET (e.g., there is no detectable acceptor fluorescence intensity). The subsequent incorporation of the fluorescence acceptor-labeled dUTP becomes possible only upon completion of the poly-G sequence. A FRET signal becomes detectable when the labeled-dUTP derivative is added after incorporation of the last labeled-dGTP (FIG.2A, lower). The rate of increase of the expected FRET signal is a measure of the efficiency of the polymerase being tested in incorporating multiple sequential labeled dNTPs. In some instances, e.g., by using template oligonucleotide sequences comprising a different number of nucleotide residues of the same type (e.g., one, two, three, four, or more than four C residues), one may evaluate polymerase processivity (i.e., the average number of nucleotides incorporated by the polymerase per association event with the template strand) and/or determine a maximum effective length of a sequence (e.g., a homopolymer sequence or a repeat sequence) for which the polymerase is capable of sustaining continuous primer extension reactions using labeled nucleotides. In some instances, the template oligonucleotide sequence used in the assay may comprise two or three different types of nucleotide residues organized in a sequential pattern (e.g., ATGATG…, etc.). In some instances, the template oligonucleotide sequence used in the assay may comprise a random stretch of two or three different types of nucleotide residues (e.g., ATTGAGTA…, etc.). In each of these cases, the 5’-terminal nucleotide residue of the template oligonucleotide is of a type that is not present in the nucleotide residues of the upstream portion of the template oligonucleotide sequence (excluding those nucleotides that may be present in a primer binding sequence located at the 3’-end of the template oligonucleotide sequence), and the labeled dNTP that is complementary to the 5’- terminal nucleotide residue of the template oligonucleotide is labeled with a fluorescent probe (e.g., a fluorescence acceptor) that is different from the fluorescent probe(s) (e.g., fluorescence donor(s)) used to label the dNTPs that are complementary to the nucleotide residues of the upstream portion of the template oligonucleotide. In some instances, 100% of the complementary dNTPs included in the reaction mixture may be labeled with a fluorescent probe (e.g., to facilitate identifying polymerases that are capable of sequential incorporation of labeled nucleotides). In some instances, less than 100% of the complementary dNTPs are labeled with a fluorescent probe (e.g., where one type of dNTP in the reaction mixture is fractionally labeled such that, e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of that dNTP is labeled) and/or where one type of dNTP in the reaction mixture is labeled and another type of dNTP is not). In some instances, fractional labeling may help alleviate fluorescence quenching and/or the difficulty of incorporating a long series of labeled nucleotides (e.g., if the goal is to find a polymerase that is optimized for <100% labeling). In some instances, observation of FRET signals may require that the fluorescence donor and fluorescence acceptor probes be positioned within about 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide residues of each other (e.g., with only 1, 2, 3, 4, 5, 6, 7, 8, or 9 intervening nucleotide residues between a nucleotide residue labeled with a donor probe and a nucleotide residue labeled with an acceptor probe) due to the inverse sixth power dependence of FRET efficiency on intermolecular separation distance (see, e.g., Sekar, et al. (2003), “Fluorescence Resonance Energy Transfer (FRET) Microscopy Imaging of Live Cell Protein Localizations”, Journal of Cell Biology 160(5):629-633). [0064] FIG. 2B provides another non-limiting example of a FRET-based polymerase assay using a template oligonucleotide sequence comprising a series of TC nucleotide repeats ending with a 5’- terminal G (note that the 3’-terminal primer binding sequence of the template oligonucleotide is not shown in this figure). As indicated in FIG.2B, the incoming labeled dNTPs used in this case were G* (a dGTP labeled with a fluorescence donor, e.g., Atto532), A* (a dATP labeled with a fluorescence donor, e.g., Atto532), and C @ (a dCTP labeled with a fluorescence acceptor, e.g., Atto633). The nucleotide incorporation reaction begins following the formation of a reaction mixture (FIG.2B, upper) comprising the template oligonucleotide sequence, primer sequence (not shown), the labeled complementary dNTPs, and the polymerase. Initially, only the labeled dATP and dGTP derivatives are incorporated by the polymerase (FIG.2B, middle). These initial incorporation events remain undetectable by FRET (e.g., there is no detectable acceptor fluorescence intensity). The subsequent incorporation of the fluorescence acceptor-labeled dCTP becomes possible only upon completion of the poly-TC sequence. A FRET signal becomes detectable when the labeled-dCTP derivative is added after incorporation of the last labeled-dATP (FIG.2B, lower). [0065] In some instances, the fluorescence intensity of the fluorescence donor and/or acceptor may be monitored as a function of time following addition of the last component (e.g., the polymerase) to a reaction mixture comprising the template oligonucleotide sequence, the primer sequence, the labeled dNTPs, and the polymerase). In some instances, the fluorescence intensity of the fluorescence donor and/or acceptor may be monitored at a specified time (e.g., an endpoint) following addition of the last component (e.g., the polymerase) to a reaction mixture comprising the template oligonucleotide sequence, the primer sequence, the labeled dNTPs, and the polymerase). In some instances, a fluorescence donor – quencher pair may be used instead of a fluorescence donor – acceptor pair (e.g., with the fluorescence quencher attached to the dNTP comprising the complementary base to the template nucleotide at the 5’ end of the homopolymer or repeat sequence), and a decrease in the fluorescence intensity of the fluorescence donor is measured as a function of time (or at a specified endpoint) following addition of the final component to the assay reaction mixture. In some instances, the methods may be used to evaluate and compare different polymerases based on their respective abilities to incorporate labeled nucleotides or their respective processivities for, e.g., homopolymer sequences. In some instances, the methods may be used to evaluate, compare, and/or select a mutant polymerase from a library of mutant polymerases based on their respective abilities to incorporate labeled nucleotides or their respective processivities for, e.g., homopolymer sequences. In some instances, template sequences may comprise a sequence of oligonucleotide residues comprising two or more types of nucleotides. In some such instances, at least one type of nucleotides in the sequence of oligonucleotide residues must comprise the first member of the fluorescence (e.g., fluorescence donor-acceptor or fluorescence donor-quencher) probe pair, and the second nucleotide comprising the second member of the fluorescence probe pair must be a different type of nucleotide than nucleotides in the sequence of oligonucleotide residues. [0066] In some instances, the methods comprise the use of three fluorophores (e.g., two fluorescence donor – acceptor pairs comprising a common member) to provide more information on the kinetics of the polymerase reaction (important, e.g., for sequencing reaction efficacy). In some instances, for example, a first fluorophore (e.g., a fluorescence donor) may be attached to a nucleotide residue at the 5’ end of the template oligonucleotide sequence, a second fluorophore which is a fluorescence acceptor for the first fluorophore (fluorescence donor) and is also a fluorescence donor for the third fluorophore is attached to the dNTPs that are complementary to the homopolymer or repeat sequence portion of the template oligonucleotide sequence, and a third fluorophore which is a fluorescence acceptor for the second fluorophore is attached to the dNTP that is complementary to the template nucleotide residue at the 5’ end of the homopolymer or repeat sequence portion of the template oligonucleotide sequence. In some instances, the fluorescence intensity of the first fluorophore, the second fluorophore, and/or the third fluorophore may be monitored as a function of time following addition of the last component (e.g., the polymerase) to a reaction mixture comprising the template oligonucleotide sequence, the primer sequence, the labeled dNTPs, and the polymerase). In some instances, the fluorescence intensity of the first fluorophore, the second fluorophore, and/or the third fluorophore may be monitored at a specified time (e.g., an endpoint) following addition of the last component (e.g., the polymerase) to a reaction mixture comprising the template oligonucleotide sequence, the primer sequence, the labeled dNTPs, and the polymerase). In some instances, a fluorescence quencher may be used instead of one of the fluorophores (e.g., instead of the third fluorophore), and a decrease in the fluorescence intensity of the first fluorophore or the second fluorophore is measured as a function of time (or at a specified endpoint) following addition of the final component to the assay reaction mixture. In some instances, the methods may be used to evaluate and compare different polymerases based on their respective abilities to incorporate labeled nucleotides or their respective processivities for, e.g., homopolymer sequences. In some instances, the methods may be used to evaluate, compare, and/or select a mutant polymerase from a library of mutant polymerases based on their respective abilities to incorporate labeled nucleotides or their respective processivities for, e.g., homopolymer sequences. [0067] In some instances of any of the assay formats described herein, the methods may be used to calculate or estimate an average nucleotide incorporation rate by determining a rate of change of the detected FRET signal and making use of the known composition and length of the template oligonucleotide sequence (e.g., the number of nucleotide residues in the template oligonucleotide sequence). In some instances of any of the assay formats described herein, the methods may be used to determine an effective maximum homopolymer length for which the polymerase is capable of efficiently incorporating labeled complementary nucleotides, or to determine an ability of the polymerase to sustain continuous primer extension reactions using labeled nucleotides, by comparing FRET signal measurements made using template oligonucleotide sequences of different lengths (e.g., comprising different numbers of nucleotide residues in the template oligonucleotide sequence). [0068] Nucleotides: As noted above, the term “nucleotide” as used herein encompasses a nucleoside triphosphate, e.g., a deoxyribonucleoside triphosphate (dNTP) or ribonucleoside triphosphate (NTP) comprising: (i) a nitrogenous base (or nucleobase) (e.g., adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U)), (ii) a 5 carbon sugar moiety (either deoxyribose or ribose, respectively); and (iii) three phosphate moieties. In some instances, a nucleotide comprises a non-natural nucleotide that comprises a non-natural (or synthetic) nucleobase (see, for example, Walsh, J. and Beuning, P. (2012), “Synthetic Nucleotides as Probes of DNA Polymerase Specificity”, J. Nucleic Acids 2012:530963). Examples of non-natural nucleobases include, but are not limited to, isocytosine bases, isoguanosine bases, methyl-substituted phenyl nucleobase analog (e.g., monomethylated, dimethylated, trimethylated, or tetramethylated benzene analogs), hydrophobic nucleobase analogs (e.g., 7-propynyl isocarbostyril nucleoside (dPICS), isocarbostyril nucleoside (ICS), 3-methylnaphthalene (3MN), or azaindole (7AI)), purine/pyrimidine mimics (e.g., a substituted azole heterocyclic carboxamide or a substituted indole scaffold), or fluorescent base analogs (e.g., 2-aminopurine (2AP) or the cytosine analogs 1,3-Diaza-2-oxophenothiazine and 1,3- Diaza-2-oxophenoxazine). In some instances, a nucleotide may further comprise a label, tag, or dye (e.g., a fluorophore or quantum dot) attached directly to the nucleotide. [0069] Linkers: In some instances, the labeled nucleotides of the present disclosure comprise a label, tag, or dye (e.g., a fluorophore or quantum dot) attached to the nucleotide via a linker moiety. In some instances, the use of a linker to attach the fluorophore or other optical label to a nucleotide may help to reduce quenching of the associated signal when performing, e.g., sequencing reactions. In some embodiments, a linker moiety comprises a cleavable moiety such as a disulfide group. In some embodiments, lengths of functional groups connecting the cleavable to the nucleotide and/or dye may vary. In some embodiments, a linker moiety further comprises a spacer moiety such as a polyhydroxyproline (hyp-n) group. Examples of suitable linker molecules include, but are not limited to, aminoethyl-SS-propionic acid (epSS), aminoethyl-SS-benzoic acid, aminohexyl-SS- propionic acid, hyp-10, and hyp-20. Labeled nucleotides comprising linkers are described in more detail in International Patent Application Publication No. WO 2020/172197, which is incorporated herein by reference in its entirety. [0070] Template oligonucleotide sequences: The template oligonucleotide sequences used in the disclosed methods comprise a primer binding site, one or more nucleotide residues (e.g., a single nucleotide residue, or one or more nucleotide residues comprising the same base or a mixture of different bases), and at least one nucleotide residue at a 5’ end of the one or more nucleotide residues that is different from the one or more nucleotide residues (e.g., at least one nucleotide residue comprising a different base from those present in the one or more nucleotide residues). In some instances, the template oligonucleotide sequence may further comprise flanking sequences of arbitrary length at either the 3’ and/or 5’ end of this basis template oligonucleotide sequence. [0071] In some instances, the number of nucleotide residues in a sequence segment comprising the one or more nucleotide residues comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 nucleotide residues. In some instances, the one or more nucleotide residues are of the same type (e.g., have the same base) and comprise a homopolymer sequence. [0072] In some instances, the one or more nucleotide residues may comprise, for example, a single nucleotide residue, a homopolymer sequence, a short tandem repeat sequence comprising a dinucleotide (e.g., AC), trinucleotide (e.g., AAC), a tetranucleotide (e.g., AACT), or longer repeat pattern for which the bases present in the one or more repeats differ. In some instances, for example, the one or more nucleotide residues may comprise a short tandem repeat sequence that has a repeat pattern of from 2 to 16 nucleotides in length and include from 2 to 10 repeats of the repeat pattern. In some instances, the one or more nucleotide residues may comprise a defined sequence, an arbitrary or random sequence, a partially-random sequence (e.g., a mix of defined and random subsequences), or non-random sequence of nucleotide residues ranging from 2 to 50 nucleotide residues in length. In some instances, the one or more nucleotide residues comprise one, two, or three types of nucleotide bases. [0073] In some instances, the overall length of the template oligonucleotide sequence, including the primer binding site, the one or more nucleotide residues, the at least one nucleotide residue at the 5’ end of the one or more nucleotide residues that is different than the one or more nucleotide residues, and any flanking sequences at either the 3’ end and/or the 5’ end of the template, may range from about 15 nucleotides in length to about 50 nucleotides in length. In some instances, the overall length of the template oligonucleotide sequence may be at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, or at least 50 nucleotides. In some instances, the overall length of the template oligonucleotide sequence may be at most 50 nucleotides, at most 45 nucleotides, at most 40 nucleotides, at most 35 nucleotides, at most 30 nucleotides, at most 25 nucleotides, at most 20 nucleotides, at most 19 nucleotides, at most 18 nucleotides, at most 17 nucleotides, at most 16 nucleotides, or at most 15 nucleotides. Any of the lower and upper values described in this paragraph may be combined to form a range included within the disclosure, for example, in some instances the overall length of the template oligonucleotide sequence may range from about 16 nucleotides to about 35 nucleotides. Those of skill in the art will recognize that the overall length of the template oligonucleotide sequence may have any value within this range, e.g., about 24 nucleotides. [0074] In some instances, the template oligonucleotide sequence comprises a label, tag, or dye (e.g., a fluorophore or quantum dot) attached directly to a nucleotide residue of the template sequence (e.g., the 3’ or 5’ nucleotide residue of the template sequence). In some instances, the template oligonucleotide sequence comprises a label, tag, or dye (e.g., a fluorophore or quantum dot) attached to a nucleotide residue of the template sequence (e.g., the 3’ or 5’ nucleotide residue of the template sequence) via a linker moiety, as described elsewhere herein. [0075] Primer sequences: The disclosed methods comprise the use of primer sequences, e.g., short oligonucleotide sequences that are complementary to and hybridize with the template oligonucleotide sequence at the primer binding site, thereby forming a double-stranded segment of nucleic acid that is recognized and bound by a polymerase to trigger incorporation of nucleotides at the 3’-OH terminus of the annealed primers. In some instances, the length of the primer sequence may range from about 8 nucleotides in length to about 30 nucleotides in length. In some instances, the length of the primer may be at least 8 nucleotides, at least 10 nucleotides, at least 12 nucleotides, at least 14 nucleotides, at least 16 nucleotides, at least 20 nucleotides, at least 22 nucleotides, at least 24 nucleotides, at least 26 nucleotides, at least 28 nucleotides, or at least 30 nucleotides. In some instances, the length of the primer may be at most 30 nucleotides, at most 28 nucleotides, at most 26 nucleotides, at most 24 nucleotides, at most 22 nucleotides, at most 20 nucleotides, at most 18 nucleotides, at most 16 nucleotides, at most 14 nucleotides, at most 12 nucleotides, at most 10 nucleotides, or at most 8 nucleotides. Any of the lower and upper values described in this paragraph may be combined to form a range included within the disclosure, for example, the length of the primer may range from about 10 nucleotides to about 22 nucleotides. Those of skill in the art will recognize that the length of the primer may have any value within this range, e.g., about 19 nucleotides. [0076] Fluorescence resonance energy (FRET) probe pairs: Any of a variety of fluorescence probe pairs (or FRET probe pairs) known to those of skill in the art may be used in implementing the methods. Examples of suitable fluorescence donor – acceptor pairs include, but are not limited to, FITC - Rhodamine, Alexa488 - Cy3, Cy3 - Cy5, Atto550–Atto647N, Alexa546 - Alexa647, Pacific Blue-Atto532, or Atto532 - Atto633. Examples of suitable fluorescence donor – quencher pairs include, but are not limited to, Quasar670 – BHQ2, CalRed – BHQ2, Quasar570 – BHQ2, TET – BHQ2, or TAMRA – BHQ2. In some preferred instances, the assay methods comprise the use of Atto 532, Atto633, and/or Pacific Blue. [0077] FIG.3 provides the structure and fluorescence absorption and emission spectra for the Atto532 fluorophore (Atto-Tec GmbH, Sigen, DE). The fluorophore has an excitation/absorbance maximum at about 532 nm, and an emission peak at about 552 nm. This fluorophore exhibits strong absorption, high fluorescence quantum yield, high thermal and photo-stability, and excellent water solubility. Due to the high degree of overlap between the Atto532 fluorescence emission spectrum and the excitation/absorbance spectrum of Atto633, the pair function as an efficient fluorescence donor – acceptor pair, with Atto532 acting as the energy donor. [0078] FIG.4 provides the structure and fluorescence absorption and emission spectra for the Atto633 fluorophore (Atto-Tec GmbH, Sigen, DE). The fluorophore has an excitation/absorbance maximum at about 630 nm, and an emission peak at about 651 nm. This fluorophore also exhibits strong absorption, high fluorescence quantum yield, high thermal and photo-stability. Again, due to the high degree of overlap between the Atto532 fluorescence emission spectrum and the excitation/absorbance spectrum of Atto633, the pair function as an efficient fluorescence donor – acceptor pair, with Atto633 acting as the energy acceptor. [0079] FIG.5 provides the structure (of the amine-reactive succinimidyl ester form) and fluorescence absorption and emission spectra for the Pacific Blue fluorophore (ThermoFisher Scientific, Waltham, MA). The fluorophore has an excitation/absorbance maximum at about 380 nm, and an emission peak at about 460 nm. Due to the high degree of overlap between the Pacific Blue fluorescence emission spectrum and the excitation/absorbance spectrum of Atto532, Pacific Blue may be used as a fluorescence energy donor, with Atto532 acting as the energy acceptor. [0080] Polymerases: The disclosed methods may be used to monitor nucleotide incorporation reactions and evaluate any of a variety of prokaryotic, eukaryotic, or bacteriophage polymerases (e.g., DNA polymerases, RNA polymerases, reverse transcriptases, etc.) known to those of skill in the art. The term “polymerizing enzyme” or “polymerase,” as used herein, generally refers to any enzyme capable of catalyzing a polymerization reaction (e.g., a nucleic acid polymerase). A polymerizing enzyme may be used to extend a nucleic acid primer paired with a template strand by incorporation of nucleotides or nucleotide analogs. A polymerizing enzyme may synthesize a new strand of DNA by extending the 3' end of an existing nucleotide chain, i.e., by adding new nucleotides that are complementary to nucleotide residues of the template strand one at a time via the creation of phosphodiester bonds. Polymerases used herein can have strand displacement activity or non-strand displacement activity. A polymerase may be a Family A polymerase or a Family B polymerase. In some cases, a polymerase is a polymerase modified to accept dideoxynucleotide triphosphates, such as for example, Taq polymerase having a 667Y mutation (see, e.g., Tabor, et al., PNAS, 1995, 92, 6339-6343, which is herein incorporated by reference in its entirety for all purposes). In some cases, a polymerase is a polymerase having a modified nucleotide binding, which may be useful for nucleic acid sequencing, with non-limiting examples that include ThermoSequenas polymerase (GE Life Sciences), AmpliTaq FS polymerase (ThermoFisher) and Sequencing Pol polymerase (Jena Bioscience). In some cases, the polymerase is genetically engineered to have discrimination against dideoxynucleotides, such as for example, Sequenase DNA polymerase (ThermoFisher). In some cases, the disclosed methods may comprise the use of a transcriptase or a ligase is used (i.e., other enzymes which catalyze the formation of a bond). [0081] Examples of prokaryotic DNA polymerases that may be assayed using the methods described herein include, but are not limited to, Pol I or fragments thereof (e.g., the Klenow fragment which lacks the 5’ → 3’ exonuclease activity of Pol I), Pol II or fragments thereof, Pol III or fragments thereof, Pol IV or fragments thereof, Pol V or fragments thereof, Pol D or fragments thereof, Taq polymerase or fragments thereof, Platinum Taq polymerase or fragments thereof, E. coli DNA polymerase I or fragments thereof, Tth polymerase or fragments thereof, Tli polymerase or fragments thereof, Pfu polymerase or fragments thereof, Pfu-turbo polymerase or fragments thereof, Pwo polymerase or fragments thereof, VENT polymerase or fragments thereof, DEEPVENT polymerase or fragments thereof, EX-Taq polymerase or fragments thereof, LA-Taq or fragments thereof, Sso polymerase or fragments thereof, Poc polymerase or fragments thereof, Pab polymerase or fragments thereof, Mth polymerase, ES4 polymerase or fragments thereof, Tru polymerase or fragments thereof, Tac polymerase or fragments thereof, Tma polymerase or fragments thereof, Tfl polymerase or fragments thereof, Bst polymerase or fragments thereof, Sac polymerase or fragments thereof, Tne polymerase or fragments thereof, Tfi polymerase or fragments thereof, Tbr polymerase or fragments thereof, Pwo polymerase or fragments thereof, Tea polymerase or fragments thereof, Tih polymerase or fragments thereof, Pyrobest polymerase or fragments thereof, KOD polymerase or fragments thereof, or any combination thereof. [0082] Examples of bacteriophage polymerases that may be assayed using the methods described herein include, but are not limited to, T7 DNA polymerase or fragments thereof, T4 DNA polymerase or fragments thereof, ĭ29 (phi29) DNA polymerase or fragments thereof, or any combination thereof. [0083] Examples of eukaryotic DNA polymerases that may be assayed using the methods described herein include, but are not limited to, Pol β or fragments thereof, Pol ı or fragments thereof, Pol λ or fragments thereof, Pol μ or fragments thereof, Pol α or fragments thereof, Pol δ or fragments thereof, Pol ε or fragments thereof, Pol η or fragments thereof, Pol 1 or fragments thereof, and Pol Κ or fragments thereof, Pol ζ or fragments thereof, Pol γ or fragments thereof, Pol θ or fragments thereof, Pol ^ or fragments thereof, Rev1 or fragments thereof, terminal deoxynucleotidyl transferase (TdT) or fragments thereof, telomerase or fragments thereof, or any combination thereof. [0084] Reverse transcriptases are enzymes used to synthesize complementary DNA (cDNA) from an RNA template. Examples of reverse transcriptases that may be assayed using the methods include, but are not limited to, the HIV-1 reverse transcriptase (from human immunodeficiency virus type 1) or fragments thereof, the M-MLV reverse transcriptase (from the Moloney murine leukemia virus) or fragments thereof, the AMV reverse transcriptase (from the avian myeloblastosis virus) or fragments thereof, and the telomerase reverse transcriptase (found in eukaryotes) or fragments thereof, human DNA pol ^ or fragments thereof, Tth polymerase or fragments thereof, E. coli DNA polymerase I or fragments thereof, Taq polymerase or fragments thereof, Bst polymerase or fragments thereof, SuperScript I or fragments thereof, SuperScript II or fragments thereof, SuperScript III or fragments thereof, SuperScript IV or fragments thereof, or any combination thereof. [0085] In some instances, the methods may be used to evaluate, rank, and/or select mutant polymerases from a library of mutant polymerases. Libraries of mutated polymerases and other proteins may be produced using any of a variety of techniques known to those of skill in the art, and may utilize random mutagenesis, site-directed mutagenesis, combinatorial mutagenesis, or insertional mutagenesis. In one approach, for example, site-directed mutagenesis may be performed using a synthesized oligonucleotide primer containing the mutation as part of a primer extension reaction with DNA polymerase, followed by cloning and expression in the desired organism, and subsequent purification of the mutant polymerase protein (see, for example, Hemsley, et al. (1989), “A Simple Method for Site-Directed Mutagenesis Using the Polymerase Chain Reaction), Nucleic Acids Research 17(16):6545). [0086] Other assay reaction components: In some instances, the assay reaction mixture used in the methods described herein comprises a variety of additional assay components. Examples include, but are not limited to, pH buffers, salts, monovalent ions, divalent ions, zwitterions, detergents and surfactants, coenzymes, inorganic or organic cofactors, and the like. [0087] Kits: Also disclosed herein are kits for performing any of the methods described herein. In some instances, a kit of the present disclosure comprises one or more template oligonucleotide sequences, one or more primers, one or more polymerases (or ligases, nucleases, etc.), two or more labeled dNTPs (e.g., Pacific Blue-labeled dNTPs, Atto532-labeled dNTPS, Atto633-labeled dNTPS, etc.), one or more buffer solutions, or any combination thereof. In some instances, a kit of the present disclosure may further comprise instructions and/or an assay protocol for performing the methods. [0088] Fluorescence detection instrumentation: In some instances, the methods may be performed using any of a variety of commercial fluorescence spectrophotometers, fluorometers, and/or microplate readers configured to detect fluorescence. In some instances, the methods may be performed using a custom-built fluorescence detection instrument. [0089] Examples of commercial fluorometers that may be suitable for use in performing the methods include, but are not limited to, Molecular Devices SpectraMax fluorescence microplate readers (Molecular Devices, San Jose, CA), the Duetta fluorescence and absorbance spectrometer (Horiba, Piscataway, NJ), the Qubit 4 Fluorometer and NanoDrop 3300 Fluorospectrometer (ThermoFisher Scientific, Waltham, MA), the Quantus™ (Promega Corp., Madison, WI). [0090] Examples of commercial microplate readers configured to detect fluorescence include, but are not limited to, the GloMax® Plate Reader (Promega Corp., Madison, WI), the Synergy LX Multi-Mode Reader (BioTek Instruments, Winooski, VT), and the Spark® and Infinite® series of multimode fluorescence plate readers (Tecan, Baldwin park, CA). [0091] In some instances, the methods may be performed using a custom-built fluorescence detection instrument comprising one or more light sources, monochromators, diffraction gratings, slits, apertures, lenses, mirrors, dichroic reflectors, dichroic filters, band-pass filters, long-pass filters, short-pass filters, interference filters, detectors (e.g., photomultipliers (PMTs), avalanche photodiodes, charge-coupled devices (CCDs), CMOS sensors, etc.), cuvette holders, flow cell holders, light tight housings, or any combination thereof. [0092] In some instances, the light source(s) of the fluorescence detection instrument, alone or in combination with one or more optical components, e.g., excitation optical filters and/or dichroic beam splitters, may produce excitation light at about 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 m, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm, 875 nm, or 900 nm. Those of skill in the art will recognize that the excitation wavelength may have any value within this range of about 350 – 900 nm, e.g., about 620 nm. [0093] In some instances, the light source(s) of the fluorescence detection instrument, alone or in combination with one or more optical components, e.g., excitation optical filters and/or dichroic beam splitters, may produce light at the specified excitation wavelength within a bandwidth of ± 2 nm, ± 5 nm, ± 10 nm, ± 20 nm, ± 40 nm, ± 80 nm, or greater. Those of skill in the art will recognize that the excitation bandwidths may have any value within this range, e.g., about ± 18 nm. [0094] In some instances, one or more detection channels of the fluorescence detection instrument comprise one or more optical components, e.g., emission optical filters and/or dichroic beam splitters, configured to collect emission light at about 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 m, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, 725 nm, 750 nm, 775 nm, 800 nm, 825 nm, 850 nm, 875 nm, or 900 nm. Those of skill in the art will recognize that the emission wavelength may have any value within this range of about 350 – 900 nm, e.g., about 825 nm. [0095] In some instances, one or more detection channels of the fluorescence detection instrument comprise one or more optical components, e.g., emission optical filters and/or dichroic beam splitters, configured to collect light at the specified emission wavelength within a bandwidth of ± 2 nm, ± 5 nm, ± 10 nm, ± 20 nm, ± 40 nm, ± 80 nm, or greater, and direct it to one or more detector(s). Those of skill in the art will recognize that the excitation bandwidths may have any value within this range of ± 2 nm to ± 80 nm, e.g., about ± 18 nm. [0096] In some instances, the methods comprise the use of a fluorescence detection instrument configured for dual wavelength excitation and/or dual wavelength emission. [0097] Polymerase library screening methods & throughput: In some instances, as noted above, the methods may be used to screen libraries of polymerases, e.g., libraries of mutant polymerases, to evaluate, rank, and/or select polymerases based on respective nucleotide incorporation rates and/or polymerase processivity. In some instances, a mutant polymerase may be selected, for example, if it is more efficient than a corresponding wild-type polymerase at incorporating nucleotides (e.g., labeled nucleotides) into a growing nucleic acid strand. In some instances, a mutant polymerase may be selected, for example, if it is less likely than a corresponding wild-type polymerase to misincorporate nucleotides (e.g., labeled nucleotides) during nucleic acid sequencing. [0098] In some instances, these methods may be implemented in a microplate format, where the microplates are configured for use in a microplate reader configured for fluorescence detection. Examples of commonly used microplate formats include 6-well, 12-well, 24-well, 48-well, 96-well, 384-well, or 1536-well microplate formats. [0099] In some instances, e.g., using the methods as deployed in a microplate format, polymerases (e.g., mutant polymerases) may be screened at a throughput rate of at least 20, at least 40, at least 60, at least 80, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 polymerases tested per hour. In some instances, the throughput for polymerase testing (or ligase or nuclease testing) may have any value within the range of values described in this paragraph. [0100] In some instances, for example, if the methods are implemented in an endpoint assay format, a FRET signal that is indicative of incorporation of labeled nucleotides into the extended primer strand may be measured at a specified time following addition of a final component (e.g., the polymerase) to the assay reaction mixture. In some instances, the FRET signal may be measured at about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, or 120 minutes after addition of the final component to the assay reaction mixture. [0101] Non-polymerase assay formats: In some instances, the methods described herein may be adapted for use to monitor other types of enzyme-catalyzed reactions, e.g., ligase reactions or nuclease reactions, where the design of the template oligonucleotide sequence may be tailored to provide information about the enzyme binding efficacy for different recognition template oligonucleotide sequences and/or enzyme processivity for different template oligonucleotide sequences. [0102] In some instances, the methods may be adapted, for example, to performing a ligase assay. Methods for monitoring a ligase reaction comprise: forming a reaction mixture comprising: (i) a first oligonucleotide sequence comprising a first nucleotide residue coupled to a first member of a fluorescence donor-acceptor pair; and (ii) a second oligonucleotide sequence comprising a second nucleotide residue coupled to a second member of a fluorescence donor-acceptor pair; and a ligase; and detecting a presence or absence of a fluorescence resonance energy transfer (FRET) signal, wherein the presence of the FRET signal indicates that the ligase has catalyzed the formation of a phosphodiester bond between a 3'-OH group at one end of the first or second oligonucleotide sequence and a 5'-phosphate group at one end of the second or first oligonucleotide sequence, respectively. In some instances, the first and second nucleotide residues are the same. In some instances, the first oligonucleotide sequence and the second oligonucleotide sequence are designed so that, upon ligation, the first nucleotide residue and the second nucleotide residue are separated by less than 8, 7, 6, 5, 4, 3, or 2 nucleotide residues. In some instances, the first and second oligonucleotide sequences may be double-stranded. In some instances, the first and second oligonucleotides comprise complementary overhanging sequences at one end. In some instances, the first and second oligonucleotide sequences may be single-stranded. In some instances, the first and second oligonucleotide sequences may be deoxyribonucleic acid (DNA) sequences, and the ligase is a DNA ligase. In some instances, the first and second oligonucleotide sequences may be ribonucleic acid (RNA) sequences, and the ligase is an RNA ligase. In some instances, the first member of the fluorescence donor-acceptor pair may be a FRET donor and the second member of the fluorescence donor-acceptor pair may be a FRET acceptor. In some instances, the fluorescence donor-acceptor pair comprises FITC - Rhodamine, Alexa488 - Cy3, Cy3 - Cy5, Atto550–Atto647N, Alexa546 - Alexa647, Pacific Blue-Atto532, or Atto532 - Atto633. In some instances, the method may further comprise determining a rate of FRET signal increase following the addition of the ligase. In some instances, the method may further comprise calculating an turnover rate for the ligase from the determined rate of FRET signal increase. In some instances, the method may be used to screen a library of mutant ligases and rank order the mutant ligases based on a magnitude of the detected FRET signal or signal increase. In some instances, the method may be used to screen a library of mutant ligases and rank order the mutant ligases based on calculated turnover rates. In some instances, the method may further comprise selecting a mutant ligase of the library based on its rank order. [0103] In some instances, the methods may be adapted, for example, to performing a nuclease assay. Methods for monitoring a nuclease reaction comprise: forming a reaction mixture comprising: a oligonucleotide sequence comprising a first nucleotide residue coupled to a first member of a fluorescence probe pair, a second nucleotide residue coupled to a second member of a fluorescence probe pair, and a short oligonucleotide sequence separating the first nucleotide residue and the second nucleotide residue; adding a nuclease to the reaction mixture; and detecting a change in a fluorescence resonance energy transfer (FRET) signal, wherein a change in the FRET signal indicates that the nuclease has cleaved a phosphodiester bond located between the first nucleotide residue and the second nucleotide residue. In some instances, the short oligonucleotide sequence may be designed such that the first nucleotide residue and the second nucleotide residue are separated by less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotide residues. In some instances, the oligonucleotide sequence comprises a double-stranded DNA sequence, a single-stranded DNA sequence, or an RNA/DNA duplex sequence. In some instances, the nuclease comprises a DNA endonuclease or an RNA endonuclease. In some instances, the short oligonucleotide sequence comprises a recognition sequence for a restriction endonuclease, and the nuclease comprises a restriction endonuclease. In some instances, the restriction endonuclease comprises a DNA restriction endonuclease or an RNA restriction endonuclease. In some instances, the fluorescence probe pair comprises a fluorescence donor-acceptor pair, and a decrease in FRET signal indicates that the nuclease has cleaved a phosphodiester bond located between the first nucleotide residue and the second nucleotide residue. In some instances, the first member of the fluorescence donor- acceptor pair is a FRET donor, and the second member of the fluorescence donor-acceptor pair is a FRET acceptor. In some instances, the fluorescence donor-acceptor pair comprises FITC - Rhodamine, Alexa488 - Cy3, Cy3 - Cy5, Atto550–Atto647N, Alexa546 - Alexa647, Pacific Blue- Atto532, or Atto532 - Atto633. In some instances, the fluorescence probe pair comprises a fluorescence donor-quencher pair, and an increase in FRET signal indicates that the nuclease has cleaved a phosphodiester bond located between the first nucleotide residue and the second nucleotide residue. In some instances, the first member of the fluorescence donor-quencher pair is a FRET donor, and the second member of the fluorescence donor-quencher pair is a FRET quencher. In some instances, the fluorescence donor-quencher pair is Quasar670 – BHQ2, CalRed – BHQ2, Quasar570 – BHQ2, TET – BHQ2, or TAMRA – BHQ2. In some instances, the method may further comprise determining a rate of FRET signal increase or decrease following the addition of the nuclease. In some instances, the method may further comprise calculating an turnover rate for the nuclease from the determined rate of FRET signal increase or decrease. In some instances, the method may be used to screen a library of mutant nucleases and rank order the mutant nucleases based on a magnitude of the detected FRET signal increase or decrease. In some instances, the method may be used to screen a library of mutant nucleases and rank order the mutant nucleases based on calculated turnover rates. In some instances, the method may further comprise selecting a mutant nuclease of the library based on its rank order. EXAMPLES Example 1 – Comparison of nucleotide incorporation rates for mutant polymerases using a FRET polymerase assay [0104] The experimental procedure for performing the FRET-based multiple nucleotide incorporation assay described herein was as follows. The template oligonucleotide used in the assay was dissolved to a final concentration of 10 nM in a buffer of 20 mM Tris-HCl, pH 8.8, 2 mM MgCl 2 , 110 mM NaCl. The two labeled complimentary nucleotides were then added to this solution, one labeled with Atto532 (the fluorescence donor) and the other labeled with Atto633 (the fluorescence acceptor), to a final concentration of 100 nM. Aliquots of this solution were then pipetted into individual wells of a 96 well plate and the plate was paced in a fluorescence microplate reader, pre-equilibrated to a temperature of 45°C. The fluorescence detection settings were set at two excitation/emission wavelength combinations, 490 nm/560 nm (to monitor Atto532 emission) and 490 nm/660 nm (to monitor Atto532 - Atto633 FRET-based emission). The fluorescence signals were monitored over the next 5-6 minutes and, once a stable baseline was achieved, the reading was interrupted to add aliquots of the polymerases being tested, typically to a final concentration of 20 – 40 nM. The fluorescence signals were then monitored for another 45-60 minutes. Typically, control wells contain either no polymerase, or only donor-labeled, or only acceptor-labeled nucleotides. [0105] FIG.6 provides a non-limiting example of results from an assay where five different polymerase mutants (Pol 57, Pol 75, Pol 92, Pol 96, and Pol 97) were tested with an oligonucleotide having the template sequence ...TTTA and the labeled complementary nucleotides. Fluorescence traces were measured as a function of time (seconds) using 490 nm excitation light while monitoring the Atto 532 - Atto633 FRET-based emission at 660 nm. As can be seen, Pol 75 and Pol 92 show the fastest nucleotide incorporation rates, followed by Pol 96, Pol 57, and Pol 97 as the slowest. [0106] FIG.7 provides another example using the template sequence ...CCCCCA. The donor- labeled and acceptor-labeled nucleotides were dGTP-Atto532 and dUTP-Atto632, respectively. Here too, the different incorporation kinetics of the polymerase mutants tested are clearly distinguishable by using 490 nm excitation light while monitoring the Atto 532 - Atto633 FRET- based emission at 660 nm. Example 2 – FRET-based polymerase assay using three fluorophores [0107] The experimental procedure for performing the FRET-based multiple nucleotide incorporation assay using three fluorophores as described herein was as follows. The template oligonucleotide (comprising a Pacific Blue fluorophore – the first fluorescence donor - attached to the 5’ end) was dissolved to a final concentration of 10 nM in a buffer of 20 mM Tris-HCl, pH 8.8, 2 mM MgCl 2 , 110 mM NaCl. The two labeled complementary nucleotides were then added to this solution, one labeled with Atto532 (the first fluorescence acceptor / second fluorescence donor) and the other labeled with Atto633 (the second fluorescence acceptor), to a final concentration of 100 - 200 nM. Aliquots of this solution were then pipetted into individual wells of a 96 well plate and the plate was paced in a fluorescence microplate reader, pre-equilibrated to a temperature of 45°C. The fluorescence detection settings were set at an excitation/emission wavelength combination of 490/660 to monitor Atto532 - Atto633 FRET-based emission. The fluorescence detection settings were set at an excitation/emission wavelength combination of 406 nm/460 nm to monitor Pacific Blue fluorescence, at 406 nm/560 nm to monitor Pacific Blue – Atto532 FRET-based emission, and at 406 nm/660 nm to monitor Pacific blue - Atto532 - Atto633 FRET-based emission. The fluorescence signals were monitored over the next 5-6 minutes and, once a stable baseline was achieved, the reading was interrupted to add aliquots of the polymerases being tested, typically to a final concentration of 20 – 40 nM. The fluorescence signals were then monitored for another 45-60 minutes. Typically, control wells contain either no polymerase, or only donor-labeled or only acceptor-labeled nucleotides. [0108] FIG.8 provides a non-limiting example of results from an assay where five different polymerase mutants were tested with an oligonucleotide having the template sequence …TTTACTTT-Fluorescein. Although the oligonucleotide in this examples was fluorescently labeled, it was not used to generate FRET signals. Rather, the two labeled complementary nucleotides, dATP-linker-Atto 532 and dUTP-linker-Atto633, were used in the same assay format as described for Example 1 (i.e., Atto532 was excited at 490 nm and Atto532 – Atto633 FRET-based emission was monitored at 660 nm). [0109] FIG.9 provides a non-limiting example of results for an assay where four different polymerase mutants were tested using an oligonucleotide having the template sequence …TTTGCTTT-Pacific Blue. In this experiment, Pacific Blue was used as the first fluorescence donor while the two labeled complementary nucleotides, dATP-linker-Atto532 (first fluorescence acceptor / second fluorescence donor) and dCTP-linker-Atto633 (second fluorescence acceptor), were used to generate a FRET-based signal monitored at 660 nm. FIG.9 illustrates the fluorescence signals recorded as a function of time for Pacific Blue fluorescence (406 nm excitation / 460 nm emission) following addition of the polymerase. The signal decreases in intensity as the Atto532- labeled nucleotides are incorporated by the polymerase, thereby providing an energy acceptor for the Pacific Blue label. FIG. 10 provides a non-limiting example of the “intermediate” fluorescence traces, i.e., the fluorescence signals recorded as a function of time for the Pacific Blue - Atto532 FRET-based signal (406 nm excitation / 560 nm emission). There is a transient increase in signal as the polymerase incorporates more of the Atto532-labeled complementary nucleotide, but the signal decreases again as the Atto633-labeled complementary nucleotide is incorporated into the template oligonucleotide sequence. FIG. 11 provides a non-limiting example of the Pacific Blue – Atto532 – Atto633 FRET-based signal (406 nm excitation / 660 nm emission). The signal rises and then plateaus as the primer extension reaction reaches completion. Note the fluorescence traces for the Pol 92 and Pol 96 polymerases. Using the original non-modified assay, These two polymerases appear to function similarly using the two fluorophore (single FRET probe pair) assay format described in Example 1, with Pol 92 perhaps functioning slightly better. However, when these polymerases were tested for use in a sequencing reaction, it was discovered that Pol 96 unexpectedly outperformed Pol 92. Pol 96 is apparently more efficient than Pol92 at incorporating labeled nucleotides. This can be observed in the “intermediate” fluorescence kinetic traces shown in FIG. 10. Pol 96 exhibited a sharper peak for each tested oligo, indicating that it more quickly incorporates the first and second labeled nucleotides. [0110] FIG. 12 provides another example of data for the two fluorophore (single FRET probe pair) assay format. In this case, three mutant polymerases were tested using an oligonucleotide having the template sequence …TTTGCTTT-Pacific Blue. Although the oligonucleotide in this examples was fluorescently labeled, it was not used to generate FRET signals. Rather, the two labeled complementary nucleotides, dATP-linker-Atto532 and dUTP-linker-Atto633, were used in the same assay format as described for Example 1 (i.e., Atto532 was excited at 490 nm and Atto532 – Atto633 FRET-based emission was monitored at 660 nm). [0111] FIG. 13 provides a non-limiting example of results for an assay where three different polymerase mutants were tested using an oligonucleotide having the template sequence …TTTGCTTT-Pacific Blue. In this experiment, Pacific Blue was again used as the first fluorescence donor while the two labeled complementary nucleotides, dATP-linker-Atto532 (first fluorescence acceptor / second fluorescence donor) and dCTP-linker-Atto633 (second fluorescence acceptor), were used to generate a FRET-based signal monitored at 660 nm. FIG.13 illustrates the fluorescence signals recorded as a function of time for Pacific Blue fluorescence (406 nm excitation / 460 nm emission) following addition of the polymerase. FIG. 14 provides a non-limiting example of the “intermediate” fluorescence traces, i.e., the fluorescence signals recorded as a function of time for the Pacific Blue - Atto532 FRET-based signal (406 nm excitation / 560 nm emission). FIG. 15 provides a non-limiting example of the Pacific Blue – Atto532 – Atto633 FRET-based signal (406 nm excitation / 660 nm emission). The fluorescence signal traces in all three figures – FIGS.13 – 15 – show similar kinetics as those illustrated in FIGS.9 – 11. Example 3 – FRET-based polymerase assay with non-homopolymer template oligo [0112] The following experiments were performed using a non-homopolymer template oligonucleotide (e.g., a template oligo that included a combination of nucleotide bases), and serve to confirm that: i) polymerases can handle incorporation of a variety of different labeled and non- labeled nucleotides, and ii) polymerases can perform nucleotide incorporation under high labeling conditions (i.e., where a larger percentage of the incorporated nucleotides are labeled). [0113] In a first experiment, a primer (5’-GTTCCTGTCCACCTCC-3’, SEQ ID NO: 4) was annealed to a template oligo having the sequence 3’-CTCTCTCTCTCTCTCTCTCTG-5’ (i.e., a (CT)10G oligo sequence, SEQ ID NO: 5) downstream from the 3’ end of the annealed primer (full length template sequence = 5’-TCAGTCTCTCTCTCTCTCTCTCTCGGAGGTGGACAGGAAC- 3’, SEQ ID NO: 6). None of the molecules (e.g., neither the primer nor the template oligo) are labeled. The primer and template oligonucleotide were dissolved to final concentrations of 15 nM and 10 nM, respectively, in a buffer of 20 mM Tris-HCl, pH 8.8, 2 mM MgCl2, 60 mM NaCl. A mutant polymerase (e.g., Pol 37; 40 nM final concentration) and an Atto 633-labeled dCTP were added to aliquots of the primer – template mixture, along with one of the following three nucleotide solutions (dNTPs at 500 nM final concentration): A. dGTP / dATP-Atto532 (G/A* mix) B. dGTP-Atto532 / dATP (G*/A mix) C. dGTP-Atto532 / dATP-Atto532 (G*/A* mix), where * indicates a labeled dNTP. [0114] Aliquots of the reaction mixture were pipetted into individual wells of a 96-well microtiter plate, and the plate was placed in a fluorescence microplate reader pre-equilibrated to a temperature of 45 °C. The mixtures were incubated in the microtiter plate at 45 o C, and the fluorescence of the mixtures was monitored using 520 nm excitation light and a 660 nm emission wavelength setting. The normalized fluorescence responses following addition of the mutant polymerase are shown in FIG. 16. These fluorescence intensity traces demonstrate the sequential incorporation of multiple labeled nucleotides by a polymerase as detected by FRET using mixtures of Atto532 (donor)- and Atto633 (acceptor)-labeled dNTPs. [0115] As can be seen in FIG.16, the 520/660 nm fluorescence intensity increases over time for all three nucleotide mixtures (the magnitude of each fluorescence signal trace was normalized by setting the starting signal to 0%, and setting the highest signal () to 100%). The rates of increase are approximately identical for the G/A* and G*/A mixtures, and is faster than for the G*/A* mixture. These observed fluorescence signal increases are indicative of successful enzymatic primer extension, where initially only Atto532-labeled nucleotides are incorporated into the complement of the poly-CT template sequence, followed eventually by the incorporation of the Atto633-labeled dCTP nucleotide opposite the G in the template. Note that a successful incorporation of the dCTP- Atto633 after one or more incorporated Atto532 labeled nucleotides is absolutely required for a 520/660 nm FRET signal to be measured. Only every other incorporated nucleotide is labeled with an Atto532 donor dye in the cases of the G/A* and G*/A mixtures, but with the G*/A* mix, the results show that a successful sequential incorporation of a total of 21 dye labeled nucleotides is possible under conditions of 100% labeling. [0116] In a second experiment, the same primer/template hybrid was used as described above, but here the 5’-terminal T residue of the template strand was labeled with a Pacific Blue dye. Only a mixture of Atto532-labeled nucleotides was used (i.e., G*/A*), and fluorescence detection was performed using 406 nm excitation (to excite Pacific Blue molecules) and 560 nm emission wavelengths. With these fluorescence detection settings, incorporation of even a single Atto532- labeled nucleotide results in an increase in the 560 nm (Atto 532 emission) signal due to resonance energy transfer from excited Pacific Blue labels (the FRET donor). This can clearly be observed in FIG. 17, which illustrates the use of this three-dye system to evaluate the sequential incorporation of multiple dye labeled nucleotides by two different polymerases (note that only the fluorescence signal arising from incorporation of Atto532 labeled nucleotides - due to resonance energy transfer from excited Pacific Blue donor molecules - is shown in this figure; fluorescence signal intensities at t = zero were set to zero). After a successful fill-in reaction to complete the (CT) 10 portion of the template sequence, incorporation of Atto633-labeled dCTP results in a decrease of the 406/560 nm fluorescence as the newly-incorporated Atto633 dye (a FRET acceptor for Atto532 donors) quenches the emission of the Atto532 label. Again, this can clearly be observed in FIG.17 - following a period of relatively small or no change in the 406/560 nm emission, there is a steady decrease of the signal as the polymerases complete the primer extension reactions. Note that the two mutant polymerases used (Pol 37 and Pol 75) show significantly different reaction kinetics, with Pol 37 being faster than Pol 75. It should be understood from the foregoing that, while particular implementations of the disclosed methods have been illustrated and described, various modifications can be made thereto and are contemplated herein. It is also not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the preferable embodiments herein are not meant to be construed in a limiting sense. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. Various modifications in form and detail of the embodiments of the invention will be apparent to a person skilled in the art. It is therefore contemplated that the invention shall also cover any such modifications, variations, and equivalents.
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