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Methods of sequencing fluorophore-quencher fret-aptamers

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Methods of sequencing fluorophore-quencher fret-aptamers


The present invention describes methods for the production and selecting of single chain (single-stranded) fluorescence resonance energy transfer (“FRET”) DNA or RNA aptamers containing fluorophores (F) and quenchers (Q) at various loci within their structures, such that when its specific matching analyte is bound and the FRET-aptamers are excited by specific wavelengths of light, the fluorescence intensity of the system is modulated (increased or decreased) in proportion to the amount of analyte added. F and Q are covalently linked to nucleotide triphosphates (NTPs), which are incorporated by various nucleic acid polymerases such as Taq polymerase during the polymerase chain reaction (PCR) and then selected by affinity chromatographic, size-exclusion or molecular sieving, and fluorescence techniques. Further separation of related FRET-aptamers can be achieved by ion-pair reverse phase high performance liquid chromatography (HPLC) or other types of chromatography. Finally, FRET-aptamer structures and the specific locations of F and Q within FRET-aptamer structures are determined by digestion with exonucleases and mass spectral nucleotide sequencing analysis. Alternatively, single DNA or RNA intrachain FRET-aptamers can be sequenced and the locations of F and Q within the structure can be determined by nanopore sequencing and the locations of F and Q within the structure can be verified by nucleic acid “combing” coupled to high-powered fluorescence microscopy.
Related Terms: Polymerase Chain Reaction Polymerase Chain Reaction (pcr)

Browse recent Pronucleotein Biotechnologies, LLC patents - ,
Inventors: John G. Bruno, Joseph Chanpong
USPTO Applicaton #: #20120270221 - Class: 435 612 (USPTO) - 10/25/12 - Class 435 


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The Patent Description & Claims data below is from USPTO Patent Application 20120270221, Methods of sequencing fluorophore-quencher fret-aptamers.

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This application is based upon and claims priority from U.S. Utility application Ser. No. 11/433,009, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Applicants\' invention relates to the field of aptamer- and nucleic acid-based diagnostics. More particularly, it relates to methods for the production and use of single chain (single-stranded) fluorescence resonance energy transfer (“FRET”) DNA or RNA aptamers containing fluorophores (“F”) and quenchers (“Q”) at various loci within their structures.

2. Background Information

FRET-aptamers are a new class of compounds, consisting in part of single-stranded oligonucleotides, desirable for their use in rapid (within minutes), one-step, homogeneous assays involving no wash steps (simple bind and detect quantitative assays). Several individuals and groups have published and patented FRET-aptamer methods for various target analytes that consist of placing the F and Q moieties either on the 5′ and 3′ ends respectively to act like a “molecular (aptamer) beacon” or placing only F in the heart of the aptamer structure to be “quenched” by another proximal F or the DNA or RNA itself. These preceding FRET-aptamer methods are all highly engineered and based on some prior knowledge of particular aptamer sequences and secondary structures, thereby enabling clues as to where F might be placed in order to optimize FRET results.

Until now, no individual or group has described a method for natural selection of single chain (intrachain) FRET-aptamers that contain both fluorophore-labeled deoxynucleotides (“F-dNTPs”) and highly efficient spectrally matched quencher (“Q-dNTP”) moieties in the heart of an aptamer binding loop or pocket by polymerase chain reaction (“PCR”). The advantage of this F and Q “doping” method is two-fold: 1) the method allows nature to take its course and select the most sensitive FRET-aptamer target interactions in solution, and 2) the positions of F and Q within the aptamer structure can be determined via exonuclease digestion of the FRET-aptamer followed by mass spectral analysis of the resulting fragments, or DNA combing and nanopore sequencing, thereby eliminating the need to “engineer” the F and Q moieties into a prospective aptamer binding pocket or loop. Sequence and mass spectral data can be used to further optimize the FRET-aptamer assay performance after natural selection as well.

Others have described nucleic acid-based “molecular beacons” that snap open upon binding to an analyte or upon hybridizing to a complementary sequence, but beacons are always end-labeled with F and Q at the 3′ and 5′ ends. FRET-aptamers may be labeled anywhere in their structure that places the F and Q within the Förster distance of approximately 60-85 Angstroms to achieve quenching prior to or after target analyte binding to the aptamer “binding pocket” (typically a “loop” in the secondary structure).

“Signaling aptamers” do not include a Q in their structures, but rather appear to rely upon the “self-quenching” of two adjacent fluorophores or the mild quenching ability of the nucleic acid itself. Both of these methods of quenching are relatively poor, because eventually F-emitted photons escape into the environment and are detectable, thereby contributing to background light and limiting the sensitivity of the FRET assay. True quenchers such as dabcyl (“D”), the “Black Hole Quenchers” (“BHQs”), and the QSY family of dyes (QSY-5, QSY-7, or QSY-9) are broad spectrum absorbing molecules that appear dark or even black in color, because they absorb many wavelengths of light and do not re-emit photons. The inclusion of a Q in the intrachain FRET-aptamer structure or the competitive aptamer FRET format, reduces background fluorescence intensity significantly, thereby increasing signal-to-noise ratios and improving assay sensitivity.

In addition to the novelty of the quencher introduction into the assay formats and advantages conferred in terms of sensitivity by cutting background fluorescence, the method of selecting single intrachain FRET-aptamers based on differential molecular weight and fluorescence intensity of the target analyte-aptamer bound subset fractions is a novel FRET-aptamer development method. The F and Q molecules used can include any number of appropriate fluorophores and quenchers as long as they are spectrally matched so the emission spectrum of F overlaps significantly (almost completely) with the absorption spectrum of Q.

SUMMARY

OF THE INVENTION

The present invention describes a single chain (single-stranded intrachain) FRET assay approach in which F and Q are incorporated into an aptamer population via their nucleotide triphosphate derivatives (for example, ALEXA FLUOR™-NTPs, CASCADE BLUE®-NTPs, CHROMATIDE®-NTPs, fluorescein-NTPs, rhodamine-NTPs, RHODAMINE GREEN™-NTPs, tetramethylrhodamine-dNTPs, OREGON GREEN®-NTPs, and TEXAS RED®-NTPs may be used to provide the fluorophores, while dabcyl-NTPs, Black Hole Quencher or BHQ™-NTPs, and QSY™ dye-NTPs may be used for the quenchers) by PCR after several rounds of selection and amplification without the F- and Q-modified bases. This process is generally referred to as “doping” with F-NTPs and Q-NTPs.

Thereafter, the single chain or intrachain FRET-aptamers in the population that still bind the intended target (after the doping process) are purified by size-exclusion chromatography columns, spin columns, gel electrophoresis or other means. Once bound and separated based on weight or other physical properties, the brightest fluorescing FRET-aptamer-target complexes are selected because they are clearly the optimal FRET candidates. The FRET-aptamers are separated from the targets by heating or chemical means (urea, formamide, etc.) and purified again by size-exclusion chromatography or other means.

These intrachain FRET-aptamers cannot be cloned for sequencing due to the need for determining the locations of F and Q in their structures. Cloning would lead to replication of the FRET-aptamer insert in the plasmid and either dilution of the desired FRET-aptamer or alteration of its F and Q locations within the aptamer. Therefore, the candidate FRET-aptamers are separated based on physical properties such as charge or weak interactions by various types of high performance liquid chromatography (“HPLC”), digested at each end with specific exonucleases (snake venom phosphodiesterase on the 3′ end and calf spleen phosphodiesterase on the 5′ end). The resulting oligonucleotide fragments, each one base shorter than the predecessor, are subjected to mass spectral analysis which can reveal the nucleotide sequences as well as the positions of F and Q within the FRET-aptamers. Once the FRET-aptamer sequence is known with the positions of F and Q, it can be further manipulated during solid-phase DNA or RNA synthesis in an attempt to make the FRET assay more sensitive and specific.

An alternative method of sequencing the FRET-aptamer to determine the absolute positions of F and Q within the nucleic acid (DNA or RNA) chain which has the advantage of sequencing individual nucleic acids is called “nanopore sequencing.” This method can be applied the FRET-aptamer to determine exactly where F and Q are because as F and Q pass through the nanopore, they will register unique electrical patterns that can be used to distinguish them from the nucleotides (adenine, cytosine, guanine, uracil or thymine). Nanopore sequencing is currently performed in two basic ways: 1) through a silicon nitride plane or membrane with nanopores created by electrons shot through the silicon nitride by an electron gun or 2) the use of natural cell membranes (e.g., phospholipid bilayers or micelles) containing natural pore-like proteins such as ion-channels or gates or bacterial alpha-hemolysin through which single-stranded DNA and RNA can pass. Electrodes are set up on either side of the membrane to measure electrical activity according to Ohm\'s Law as the DNA passes through the membrane and this method could be applied to sequencing of single DNA or RNA molecules with additional information on which bases or nucleotides had F and Q covalently conjugated to them. The nucleic acid can be pushed or pulled through a nanopore by the application of positive or negative fluid pressure or electrophoresis, etc. While the electrical properties of nucleotides, F and Q are currently used to identify these components as they pass through the membrane, this does not preclude the use of other physical means such as optics (fluorescence intensity, fluorescence lifetime analysis, absorbance, or circular dichroism, etc.) to detect and identify specific nucleotides, as well as F\'s and Q\'s attached to nucleotides, as they pass through the membrane holes.

A second method, referred to as “DNA combing” (RNA can be combed as well) could be used to linearize convoluted, folded or globular nucleic acids and “comb” them out into single lines much like matted hair is combed out into individual linear strands by a comb or hair brush. One can actually image individual fluorescent DNA or RNA molecules that have been combed out on glass, silicon, of plastic microscope slides or other planar substrates using a high-powered fluorescence or optical microscope and determine the relative position of F from the 3′ or 5′ ends, thereby enabling determination of which base or nucleotide to which F is attached. It is envisioned that similar absorbance or circular dichroism or optical polarization techniques can be devised to determine which bases quenchers are attached to relative to the 3′ or 5′ ends for verification of nanopore sequencing data of intrachain FRET-aptamers.

There are a number of uses of the single-chain FRET-aptamers developed by the present invention, including quantifiable fluorescence assays for small molecules including pesticides, natural and synthetic amino acids and their derivatives (e.g., histidine, histamine, homocysteine, DOPA, melatonin, nitrotyrosine, etc.), short chain proteolysis products such as cadaverine, putrescine, the polyamines spermine and spermidine, nitrogen bases of DNA or RNA, nucleosides, nucleotides, and their cyclical isoforms (e.g., cAMP and cGMP), cellular metabolites (e.g., urea, uric acid), pharmaceuticals (therapeutic drugs), drugs of abuse (e.g., narcotics, hallucinogens, gamma-hydroxybutyrate, etc.), cellular mediators (e.g., cytokines, chemokines, immune modulators, neural modulators, inflammatory modulators such as prostaglandins, etc.), or their metabolites, explosives (e.g., trinitrotoluene) and their breakdown products or byproducts, peptides and their derivatives. Other uses of the single-chain FRET-aptamers include use in quantifiable fluorescence assays for macromolecules including proteins such as biotoxins including botulinum toxins, Shiga toxins (See FIG. 2), staphylococcal enterotoxins, other bacterial toxins, prions such as bovine spongiform encephalopathies (“BSEs”) and transmissible spongiform encephalopathies (“TSEs”), glycoproteins, lipids, glycolipids, triglycerides, nucleic acids, polysaccharides, lipopolysaccharides, etc. The single-chain FRET-aptamers may also be used in quantifiable fluorescence assays for subcellular and whole cell targets including subcellular organelles such as ribosomes, Golgi apparatus, vesicles, microfilaments, microtubules, etc., viruses, virions, rickettsiae, bacteria, protozoa, plankton, parasites such as Cryptosporidium species, Giardia species, mammalian cells such as various classes of leukocytes, neurons, stem cells, cancer cells, etc.

The use of unlabeled aptamer sequence information and secondary stem-loop structures may aid in the determination and engineered optimization of F or Q placement within the aptamer structure to maximize FRET assay sensitivity and specificity. As described and claimed herein, “within” and “interior” are defined as directing F or Q placement, or labeling, interior to, or between, the ends of the aptamer; the ends of the aptamer being the 3′ and 5′ ends. Although, an anticipated step in the present method of natural selection of F- and/or Q-labeled aptamers to form solution phase interactions with their target analytes, additional sequence and secondary structure information can be used to confirm and enhance F and Q placement to optimize assay performance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is a schematic illustration of the single, chain (intrachain) FRET-aptamer selection method.

FIG. 2. is a bar graph showing toxin concentration mapped with fluorescence intensity and illustrating a “lights off” FRET with shiga-like toxin 1 and round 5 aptamers.

FIG. 3. is a schematic illustration that illustrates a comparison of possible nucleic acid FRET assay formats.

FIG. 4. illustrates sample aptamer sequences.

FIGS. 4A-4B. are schematic illustrations of the secondary structures of selected aptamer sequences shown in FIG. 4.

FIG. 5. is a line graph correlating absorbance with BoNT A concentration.

FIGS. 6A-6B. are line graphs mapping fluorescence intensity against time.

FIG. 7. illustrates how and where nanopore DNA or RNA sequencing and nucleic acid “combing” fit into the overall intrachain FRET-aptamer process depicted in FIG. 1.

FIG. 8. illustrates how nanopore nucleic acid sequencing works to sequence the nucleotides and determine the positions of F and Q on a single intrachain-FRET aptamer by electrical measurements.

FIGS. 9A-9D. illustrate variations on performance of the combing process on top of an inverted fluorescence microscope objective.

FIG. 10. illustrates how DNA or RNA combing could linearize and determine the position of F relative to one end of the aptamer.

DETAILED DESCRIPTION

OF THE PREFERRED EMBODIMENT

Referring to the figures, FIG. 1. illustrates a single chain (intrachain) FRET-aptamer selection method. This method consists of several steps. First the random DNA library of oligonucleotides (randomized region of 20 or more bases flanked by known primer regions) is “doped” with F-dNTPs Q-dNTPs by the PCR (10). The F and Q doped library is then exposed to a protein or other target molecule (12). Some members of the doped library will bind to the target protein (14).

If the target molecule is a larger water-soluble molecule such as a protein, glycoprotein, or other water soluble macromolecule, then exposure of the nascent F-labeled and Q-labeled DNA or RNA random library to the free target analyte is done in solution. If the target is a soluble protein or other larger water-soluble molecule, then the optimal FRET-aptamer-target complexes are separated by size-exclusion chromatography. The FRET-aptamer-target complex population of molecules is the heaviest subset in solution and will emerge from an appropriately chosen (based on known fractionation data) size-exclusion column first, followed by unbound FRET-aptamers and unbound proteins or other targets. Among the subset of analyte-bound aptamers there will be heterogeneity in the numbers of F-dNTPs and Q-NTPs that are incorporated as well as nucleotide sequence differences, which will again effect the mass, electrical charge, and weak interaction capabilities (e.g., hydrophobicity and hydrophilicity) of each analyte-aptamer complex. These differences in physical properties of the aptamer-analyte complexes can then be used to separate out or partition the bound from unbound analyte-aptamer complexes.

If the target is a small molecule (generally defined as a molecule with molecular weight of ≦1,000 Daltons), then exposure of the nascent F-labeled and Q-labeled DNA or RNA random library to the target is done by immobilizing the target. The small molecule can be immobilized on a column, membrane, plastic or glass bead, magnetic bead, or other matrix. If no functional group is available on the small molecule for immobilization, the target can be immobilized by the Mannich reaction (formaldehyde-based condensation reaction) on a device similar to a PHARMALINK™ column. Elution of bound DNA from the small molecule affinity column, membrane, beads or other matrix by use of 0.2-3.0M sodium acetate at a pH ranging between 3 and 7, although the optimal pH is approximately 5.2.

These complexes can be separated from the non-binding doped DNA molecules by running the aptamer-protein aggregates (or selected aptamers-protein aggregates) through a size-exclusion column, by means of size-exclusion chromatography using Sephadex™ or other gel materials in the column (16). Since they vary in weight due to variations in aptamers sequences and degree of labeling, they can be separated into fractions with different fluorescence intensities. Purification methods such as capillary electrophoresis or preparative gel electrophoresis are possible as well. Small volume fractions (≦1 mL) can be collected from the column and analyzed for absorbance at 260 nm and 280 nm which are characteristic wavelengths for DNA, RNA and proteins. The heaviest materials come through a size-exclusion column first. Therefore, the DNA-protein complexes or RNA-protein complexes will come out of the column before either the DNA or protein alone when using an appropriate grade of column matrix material (e.g., various grades of Sephadex, Superdex, Sepharose, or Sephacryl, etc.).

Means of separating FRET-aptamer-target complexes from solution by alternate techniques (other than size-exclusion chromatography) include, without limitation, various molecular weight cut off (“MWCO”) spin columns, dialysis, gel electrophoresis, thin layer chromatography (“TLC”), and differential centrifugation or ultracentrifugation using density gradient materials.

A preferred FRET-aptamer is selected. A preferred FRET-aptamer is one that exhibits a measurable change in fluorescence between the fluorescence of said FRET-aptamer when it is not bound to said target molecule compared to the fluorescence of said FRET-aptamer when it is bound to said target molecule.

The optimal (most sensitive or highest signal to noise ratio) FRET-aptamers among the bound class of FRET-aptamer-target complexes are identified by assessment of fluorescence intensity for various fractions of the FRET-aptamer-target class. The separated DNA-protein complexes will exhibit the highest absorbance at established wavelengths, such as 260 nm and 280 nm similar to that graphed in FIG. 1 (18). The fractions showing the highest absorbance at the given wavelengths, such as 260 nm and 280 nm, are then further analyzed for fluorescence and those fractions exhibiting the greatest fluorescence are selected for separation and sequencing.

These similar FRET-aptamers may be further separated using techniques such as ion pair reverse-phase HPLC, ion-exchange chromatography (“IEC”, either low pressure or HPLC versions of IEC), TLC, capillary electrophoresis, or similar techniques.

The final FRET-aptamers are able to act as one-step “lights on” or “lights off” binding and detection components in assays.

Intrachain FRET-aptamers that are to be used in assays with long shelf-lives may be lyophilized (freeze dried) and reconstituted as needed.

FIG. 2. is a bar graph showing toxin concentration mapped with fluorescence intensity and illustrating a “lights off” FRET response with shiga-like toxin 1 and round 5 aptamers. If the fluorescence intensity of the DNA aptamers is correlated to the concentration of the surface protein and the fluorescence intensity decreases as a function of increasing analyte concentration, then it is referred to as a “lights off” assay. If the fluorescence intensity increases as a function of increasing analyte concentration, then it is referred to as a “lights on” assay. Intrachain FRET-aptamer assay data are shown for detection of E. coli shiga-like toxin I protein resulting in a “lights off” FRET reaction as a function of toxin concentration. Fluorescence readings were obtained within five minutes of toxin addition.

FIG. 3. illustrates a comparison of possible nucleic acid FRET assay formats. Upper left is a molecular beacon (30) which may or may not be an aptamer, but is typically a short oligonucleotide used to hybridize to other DNA or RNA molecules and exhibit FRET upon hybridizing. Molecular beacons are only labeled with F and Q at the ends of the DNA molecule. Lower left is a signaling aptamer (32), which does not contain a quencher molecule, but relies upon fluorophore self-quenching or weak intrinsic quenching capacity of the DNA or RNA to achieve limited FRET. Upper right is an intrachain FRET-aptamer (34) containing F and Q molecules built into the interior structure of the aptamer. Intrachain FRET-aptamers are naturally selected and characterized by the processes described herein. Lower right shows a competitive aptamer FRET (36) motif in which the aptamer contains either F or Q and the target molecule (38) is labeled with the complementary F or Q. Introduction of unlabeled target molecules (40) then shifts the equilibrium so that some labeled target molecules (38) are liberated from the labeled aptamer (36) and modulate the fluorescence level of the solution up or down thereby achieving FRET. A target analyte (40) is either unlabeled or labeled with a quencher (Q). F and Q can be switched or swapped from placement in the aptamer (36) to placement in the target analyte (40) and vice versa.

FIG. 4 illustrates sample aptamer sequences for botulinum toxin A (BoNT A) in which all sequences are arranged 5′ to 3′ from left to right. The actual degenerate (randomized) aptamer regions are bolded. Clear consensus regions are bolded and italicized. Flanking sequences match with the primers used in the PCR scheme or the complementary strand primer sequences except in highlighted cases. Most sequences end in a 3′ A (added by Taq, underlined). Aptamer sequences that bind and inhibit the action of botulinum A (BoNT A) 150 kD holotoxin and the 50 kD enzymatic light chain or subunit of BoNT A, and which may be useful in single chain FRET-aptamer or competitive aptamer-FRET assays for detection and quantification of BoNT A.

FIGS. 4A-4D. illustrate secondary structures of selected aptamer sequences listed in FIG. 4. Various botulinum A (BoNT A) DNA aptamer secondary (two dimensional) stem-loop structures that bind the holotoxin (FIG. 4A, which exemplifies a sequence that occurred in four different clones), and bind and inhibit (See FIGS. 6A and 6B) the small (50 kD) enzymatic subunit (FIGS. 4B-4D, showing the secondary structures for three different sequences that produced similar secondary structures).

FIG. 5. is a line graph correlating absorbance with BoNT A concentration. It illustrates that aptamer-peroxidase colorimetric plate binding assay results using polyclonal BoNT A aptamers and BoNT A holotoxin. Two different trials or runs are shown. Absorbance was quantified at 405 nm using standard ABTS substrate and hydrogen peroxide activator reagents. The curves illustrate binding and sensitive detection of BoNT A by the aptamers at a level of at least 12.5 ng/mL.

FIGS. 6A-6B. are line graphs mapping the fluorescence intensity of the DNA aptamers such as those shown in FIGS. 4A-4D against time in minutes. DNA aptamers, such as those shown in FIGS. 4A-4D, bind and inhibit the enzymatic activity of BoNT A. Here the inhibition of BoNT A\'s enzymatic activity is further proof of tight aptamer binding to the toxin. FIG. 6A shows assay results using the BoNT A holotoxin and FIG. 6B shows results using the isolated 50 kD enzymatic subunit of BoNT A. The positive control line shows greater fluorescence intensity over time for the uninhibited BoNT SNAPTIDE™ assay and the “Test with Aptamer” line shows consistent suppression of the fluorescence intensity of the SNAPTIDE™ assay further proving aptamer binding and aptamer-mediated inhibition of BoNT A enzymatic activity.

FIG. 7. is an extension of FIG. 1 illustrating how and where the alternative processes of nanopore sequencing and nucleic acid combing verification fit into the overall intrachain FRET-aptamer process for dealing with a single molecule of user chosen “preferred” FRET-aptamers or a very small number (e.g., less than ten) of preferred intrachain FRET-aptamers. FIG. 7 is a further indication of how and where nanopore DNA or RNA sequencing and nucleic acid “combing” fit into the overall intrachain FRET-aptamer process depicted in FIG. 1 to aid in determining where F and Q are in intrachain FRET-aptamers. In the case of a “lights on” FRET assay, “preferred” means the FRET-aptamer and target complex produces an increased emission of detectable fluorescence after the FRET-aptamer is bound to the target molecule as compared to the amount of detectable fluorescence emitted before the two are bound. And, in the case of a “lights off” FRET assay, “preferred” means the FRET-aptamer and target complex produces a decreased emission of detectable fluorescence after the FRET-aptamer is bound to the target molecule as compared to the amount of detectable fluorescence emitted before the two are bound. However, it should be noted that “preferred” refers to a FRET-aptamer with a detectable change in detectable fluorescence (either increased or decreased as per the type of assay) as chosen by the user. “Preferred” does not necessarily refer to the single FRET-aptamer that produces most or least detectable fluorescence after binding with a target molecule, nor the greatest change in detectable fluorescence after binding with a target molecule. Thus, there may be multiple “preferred” FRET-aptamers in a population, or specific to a target.

One weakness or obstacle to industrial implementation of the intrachain FRET-aptamer approach is that selecting the “preferred” FRET-aptamer or FRET-aptamers from the remaining aptamer population from the pool by chromatography as shown in FIG. 7, generally leaves only a very small amount of FRET-aptamer DNA. In theory, the number of “preferred” FRET-aptamer molecules could be as few as one F- and Q-labeled DNA molecule which would be difficult or even impossible to sequence by the combination of 3′ and 5′ exonuclease degradation (base by base or nucleotide by nucleotide cleavage) followed by mass spectral analyses.

Once selected, the FRET-aptamer may be separated from the target molecule by heating or chemical means. After the FRET-aptamer-target molecule dissociation, there may only be a few or even a single selected FRET-aptamer remaining from the original aptamer population. If the user determines the “preferred” FRET-aptamer to be one that has been found in a very small amount, sequencing individual DNA FRET-aptamers or small numbers of FRET-aptamers is still possible via 1) nanopore DNA or RNA sequencing, or 2) DNA or RNA “combing” combined with fluorescence microscopy to determine or verify the relative positions of F and Q within the FRET aptamer\'s structure after the aptamer is linearized or “combed.”



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stats Patent Info
Application #
US 20120270221 A1
Publish Date
10/25/2012
Document #
File Date
09/01/2014
USPTO Class
Other USPTO Classes
International Class
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Polymerase Chain Reaction
Polymerase Chain Reaction (pcr)


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