Systems and methods of analyzing nucleic acid polymers and related components

A method of determining the sequence of a nucleic acid polymer, comprising

• synthesizing the nucleic acid polymer and/or its complementary strand using labeled ribonucleotide and/or deoxyribonucleotide triphosphates, and
• identifying labeled ribonucleotides and/or deoxyribonucleotides in the nucleic acid polymer and/or its complementary strand using a particle beam,
• wherein the labeled ribonucleotides and/or deoxyribonucleotides, when incorporated in the nucleic acid polymer and/or its complementary strand are identifiable using the particle beam,
• optionally wherein the nucleic acid polymer and/or the complementary strand is DNA or RNA;
• optionally wherein the nucleic acid polymer and/or its complementary strand is synthesized by a nucleic acid polymerase enzyme, preferably using polymerase chain reaction (PCR);
• optionally wherein the labels are specific for each type of nucleotide, preferably wherein the labels are incorporated in the ribonucleotide and/or deoxyribonucleotide triphosphates used in synthesis of the nucleic acid polymer and/or the complementary strand, or wherein nucleotide specific labels are bonded to the nucleotides of the nucleic acid polymer and/or the complementary strand after incorporation of the ribonucleotide and/or deoxyribonucleotide triphosphates into the nucleic acid polymer and/or the complementary strand;
• preferably wherein the labels comprise one or more atoms, more preferably wherein the atoms have an atomic number, alone or in aggregate, of greater than 55 or wherein the atoms have an atomic number, alone or in aggregate, of less than or equal to 55, and/or wherein the atoms are halogen atoms;
• preferably wherein the labels comprise three or fewer atoms, more preferably wherein the labels comprise a single atom;
• and/or
• optionally wherein the step of identifying the labeled ribonucleotides and/or deoxyribonucleotides comprises generating a particle beam, exposing the nucleic acid polymer and the complementary strand to the particle beam, and identifying the ribonucleotides and/or deoxyribonucleotides due to characteristic changes to the particle beam, preferably wherein the step of detecting the ribonucleotides and/or deoxyribonucleotides comprises detecting characteristic changes to the particle beam.

A method of determining the sequence of a nucleic acid polymer, comprising

• synthesizing a complementary strand of the nucleic acid polymer using labeled ribonucleotide triphosphates or deoxyribonucleotide triphosphates,
• attaching the nucleic acid polymer and/or the complementary strand to a substrate,
• substantially straightening the nucleic acid polymer and/or the complementary strand using molecular combing,
• generating a particle beam,
• exposing the nucleic acid polymer and the complementary strand to the particle beam through the complementary strand on the substrate, and
• interpreting changes in the particle beam resulting from interactions with the nucleotides to detect the labeled nucleotides in the complementary strand, whereby the sequence of a nucleic acid polymer is determined.

A method of detecting the presence and/or identifying a nucleic acid polymer, comprising

• forming a complementary strand of the nucleic acid polymer,
• attaching the complementary strand and, optionally, the nucleic acid polymer to a substrate, and
• detecting the presence and/or identifying the complementary strand and/or the nucleic acid polymer using a particle beam;
• optionally wherein the step of identifying comprises measuring the length or determining at least a partial sequence of the complementary strand and/or the nucleic acid polymer;
• optionally wherein the nucleic acid polymer and/or its complementary strand is DNA or RNA;
• optionally wherein the nucleic acid polymer and/or its complementary strand is formed by a nucleic acid polymerase enzyme, preferably wherein the nucleic acid polymerase enzyme is a DNA-dependent DNA polymerase, a RNA-dependent DNA polymerase or a RNA-dependent RNA polymerase;
• optionally wherein the nucleotides of the nucleic acid polymer and/or the complementary strand are modified to include labels,
• preferably wherein the labels are specific for each type of nucleotide, more preferably wherein nucleotide specific labels are incorporated in the nucleic acid polymer and/or the complementary strand during formation of the nucleic acid polymer and/or the complementary strand or wherein nucleotide specific labels are bonded to the nucleotides of the nucleic acid polymer and/or the complementary strand after formation of the nucleic acid polymer and/or the complementary strand, and/or
• preferably wherein the labels comprise one or more atoms, more preferably wherein the atoms have an atomic number, alone or in aggregate, of greater than 55 or wherein the atoms are have an atomic number, alone or in aggregate, of less than or equal to 55, optionally wherein the atoms are halogen atoms, and/or
• preferably wherein the labels comprise three or fewer atoms, more preferably wherein the labels comprise a single atom;
• and/or
• the method optionally further comprising quantifying the amount of the complementary strand and/or the nucleic acid polymer.

A device comprising

• a substrate that is substantially transparent to a particle beam, and nucleic acid polymer binding sites on a surface of the substrates optionally wherein the substrate is substantially transparent to an ion beam;
• optionally wherein the substrate comprises a carbon thin film;
• optionally further comprising a support that is substantially transparent to a particle beam;
• optionally wherein the substrate is less than 5 nm thick, preferably wherein the substrate is less than 2 nm thick, more preferably wherein the substrate is less than 1.5 nm thick, still more preferably wherein the substrate is less than 1.1 nm thick;
• optionally wherein the nucleic acid polymer binding sites are in a grid pattern;
• optionally wherein the nucleic acid polymer binding sites are sequence specific, preferably wherein the sequence specific nucleic acid polymer binding sites are olizonucleotides:
• optionally wherein the nucleic acid polymer binding sites are not sequence specific;
• and/or
• optionally further comprising one or more nucleic acid polymers affixed to the nucleic acid polymer binding sites, preferably wherein the one or more nucleic acid polymers are modified to include labels.

A method for making a device comprising

• obtaining a substrate that is substantially transparent to a particle beam, and
• forming nucleic acid polymer binding sites on a surface of the substrates
• optionally wherein the substrate is substantially transparent to an ion beam,
• optionally wherein the binding sites are formed at predetermined positions on the surface of the substrate;
• optionally wherein the substrate comprises a carbon thin film;
• optionally further comprising attaching to the substrate a support that is substantially transparent to a particle beam;
• optionally wherein the substrate is less than 5 nm thick, preferably wherein the substrate is less than 2 nm thick, more preferably wherein the substrate is less than 1.5 nm thick, still more preferably wherein the substrate is less than 1.1 nm thick;
• optionally wherein the nucleic acid polymer binding sites are formed in a grid pattern;
• optionally wherein the nucleic acid polymer binding sites are sequence specific, preferably wherein the sequence specific nucleic acid polymer binding sites are oligonucleotides;
• optionally wherein the nucleic acid polymer binding sites are not sequence specific;
• and/or
• optionally further comprising affixing one or more nucleic acid polymers to the nucleic acid polymer binding sites, preferably wherein the one or more nucleic acid polymers are modified to include labels.

A system designed to detect the presence of, determine the sequence of and/or identify a nucleic acid polymer, comprising:

• a sample chamber;
• a particle beam generator associated with the chamber;
• a sample comprising a labeled complementary strand of a nucleic acid polymer, wherein the sample, when positioned in the chamber, is exposed to a particle beam generated by the particle beam generator resulting in an interaction between the particle beam and the complementary strand;
• and
• a detector constructed and arranged to collect particle beam species after the interactions
• optionally further comprising a data analysis module operative to receive and analyze signals from the detector, preferably wherein the data analysis module is operative to analyze signals related to absorbance, reflection, deflection, energy or direction and/or wherein the data analysis module is operative to analyze pattern recognition techniques to analyze the signals and/or further comprising a user interface operative to control a display of information received and/or generated by the data analysis module;
• optionally wherein the particle beam generator is an ion beam generator;
• and/or
• optionally further comprising a feedback module designed to calibrate the system based on nucleic acid polymer data.

A system designed to detect the presence of, determine the sequence of and/or identify a nucleic acid polymer, comprising:

• a sample chamber;
• a particle beam generator associated with the chamber;
• a detector constructed and arranged to collect particle beam species after interaction between the particle beam and a sample comprising the nucleic acid polymer and/or a complementary strand of the nucleic acid polymer;
• a data analysis module designed to analyze signals related to the particle beam species to determine information related to the nucleic acid polymer;
• and
• a feedback module designed to calibrate the system based on the information;
• optionally wherein the sample comprises a labeled complementary strand of a nucleic acid polymer;
• optionally wherein the feedback module is designed to calibrate the system based on a base-base distance of the nucleic acid polymer;
• and/or
• optionally wherein the feedback module is designed to calibrate the system based on known geometries of the nucleic acid polymer.

A method for calibrating a particle beam instrument, comprising acquiring data related to a nucleic acid polymer;

• and
• calibrating the instrument based on the data;
• optionally wherein the data is related to a base-base distance of the nucleic acid polymer;
• and/or
• optionally wherein the calibrating comprises calibrating the instrument based on known geometries of the nucleic acid polymer.

A system for detecting, sequencing and/or identifying a nucleic acid polymer based on particle beam species detected by a detector, the particle beam species resulting from exposure of a sample comprising a nucleic acid polymer and/or its complementary strand to a particle beam, the system comprising:

• a data analysis module operative to receive one or more signals from the detector, the one or more signals representing the particle beam species, and to detect, sequence and/or identify the nucleic acid polymer and/or its complementary strand comprised in the sample based at least in part on the received one or more signals;
• optionally wherein the nucleic acid polymer and/or its complementary strand is labeled;
• optionally wherein the particle beam species has one or more of the following properties, absorbance, reflection, deflection, energy and direction, and wherein the data analysis module is operative to analyze the one or more signals to determine values of the one or more properties;
• optionally wherein the data analysis module is operative to access a data resource comprising nucleic acid polymer information, the data resource including a data structure having a plurality of entries, each entry specifying information about a respective nucleic acid polymer sequence, preferably wherein the data analysis module is operative to partially sequence the nucleic acid polymer based on the one or more signals, the data analysis module further comprising:
• a combining module to combine the partial sequence with sequencing information of the nucleic acid polymer accessed from the data resource;
• optionally wherein the data analysis module comprises a comparison module operative to compare information determined from the one or more signals to the information specified by one or more of the data structure entries, preferably wherein the comparison module is operative to use pattern recognition techniques to compare the information determined from the one or more signals to the information specified by the one or more the data structure entries,
• optionally wherein the data analysis module comprises a user interface module to display information received and/or generated by the data analysis module to a user;
• optionally wherein the particle beam to which the sample is exposed is generated by a particle beam generator, and wherein the data analysis module comprises a feedback module operative to provide one or more feedback signals to the particle beam generator and/or the detector, the one or more feedback signals specifying information determined at least in part from the one or more signals received from the detector, preferably wherein the one or more feedback signals comprise information for calibrating the particle beam generator, more preferably wherein the feedback module is operative to generate the one or more feedback signals based at least in part on known geometries of the nucleic acid polymer, more preferably wherein the data analysis module comprises a storage module operative to store information received and/or generated by the data analysis module on a computer-readable medium;
• and/or
• optionally wherein the sample comprises a plurality of molecules of a same nucleic acid polymer and/or its complementary strand, and a plurality of particle beam species results from exposure of the plurality of molecules of the sample to the particle beam, the one or more signals representing the plurality of particle beam species wherein the data analysis module is operative to partially sequence the nucleic acid polymer based on a first of the plurality of molecules to produce a first partial sequence, and to partially sequence the nucleic acid polymer based on a second of the plurality of molecules to produce a second partial sequence, and wherein the data processing module further comprises a combining module to combine the first and second partial sequences.

A computer-readable medium having computer-readable signals stored thereon that define instructions that, as a result of being executed by a computer, control the computer to perform a process of detecting, sequencing and/or identifying a nucleic acid polymer based on particle beam species detected by a detector, the particle beam species resulting from exposure of a sample comprising a nucleic acid polymer and/or its complementary strand to a particle beam, the process comprising:

• receiving one or more signals from the detector, the one or more signals representing the particle beam species;
• and
• detecting, sequencing and/or identifying the nucleic acid polymer and/or its complementary strand comprised in the sample based at least in part on the received one or more signals;
• optionally wherein the nucleic acid polymer and/or its complementary strand is labeled;
• optionally wherein the particle beam species has one or more of the following properties;
• absorbance reflection deflection, energy and direction and wherein the act of detecting sequencing and/or identifying comprises analyzing the one or more signals to determine values of the one or more properties;
• optionally wherein the act of detecting, sequencing and/or identifying comprises accessing a data resource comprising nucleic acid polymer information, the data resource including a data structure having a plurality of entries, each entry specifying information about a respective nucleic acid polymer sequence, preferably wherein the act of detecting, sequencing and/or identifying comprises partially sequencing the nucleic acid polymer based on the one or more signals to produce a partial sequence;
• accessing partial sequence information of the nucleic acid polymer from the data resource;
• and combining the partial sequence with the partial sequence information, preferably wherein the act of detecting, sequencing and/or identifying comprises comparing information determined from the one or more signals to the information specified by one or more of the entries, more preferably wherein the act of detecting, sequencing and/or identifying comprises using pattern recognition techniques to compare the information determined from the one or more signals to the information specified by the one or more entries;
• optionally wherein the process further comprises displaying information determined from the one or more received signals to a user;
• optionally wherein the particle beam to which the sample is exposed is generated by a particle beam generator, and wherein the process further comprises providing one or more feedback signals to the particle beam generator and/or the detector, the one or more feedback signals specifying information determined at least in part from the one or more signals received from the detector, preferably wherein the act of providing comprises providing one or more feedback signals that comprise information for calibrating the particle beam generator, more preferably wherein the process further comprises generating the one or more feedback signals based at least in part on known geometries of the nucleic acid polymer;
• optionally wherein the process further comprises storing information determined from the one or more signals on a computer-readable medium;
• and/or
• optionally wherein the sample comprises a plurality of molecules of a same nucleic acid polymer and/or its complementary strand, and a plurality of particle beam species result from exposure of the plurality of molecules of the sample to the particle beam, the one or more signals representing the plurality of particle beam species, and wherein the act of detecting, sequencing and/or identifying comprises:
• partially sequencing the nucleic acid polymer based on a first of the plurality of molecules to produce a first partial sequence;
• partially sequencing the nucleic acid polymer based on a second of the plurality of molecules to produce a second partial sequence;
• and combining the first and second partial sequences.