Enzymatic DNA repair

Abstract

One or more enzymes are used to repair the damage in the synthetic DNA molecules encoding digital information. The enzyme is included in a repair mixture, which comprises one or more of the following: DNA polymerase, DNA ligase, T4 endonuclease, endonuclease IV, endonuclease VIII and uracil glycosylase. The repair mixture can also comprise one or more of the following: a buffer solution, oxidized nicotinamide adenine dinucleotide (NAD+) and deoxyribonucleoside triphosphates (dNTPs). The synthetic DNA molecules are hatched for about four hours using the repair mixture. The use of the repair solution allows the recovery of the digital information from the damaged DNA molecules.

Description

Background Art

[0001] Synthetic deoxyribonucleic acid (DNA) has the purpose of being used as the storage medium for digital information.DNA can store digital information with higher density and longer life span than other storage media.However, DNA is susceptible to damage from various sources, such as ultraviolet (UV) radiation, hydrolysis, thermal damage, toxins, mutagenic chemicals and viruses.Damage to the DNA molecule itself may make it more difficult or impossible to recover the digital information stored in the nucleotide base sequence.Storage conditions and storage time length can affect the type and degree of damage.For extremely long-term storage, such as thousands of years, DNA is also likely to suffer damage even under the most protective storage conditions.For short-term storage, the ability to repair errors can make less robust storage options feasible.

[0002] Technology that helps accurately recover digital information encoded in damaged DNA could increase the usability of DNA as a storage medium for digital information. Summary of the invention

[0003] The present disclosure provides techniques for repairing DNA to improve the recovery of digital information stored in DNA nucleotide sequences. DNA is repaired by exposure to a repair mixture comprising one or more enzymes, such as DNA polymerase, DNA ligase, and (multiple) endonucleases. Enzymatic repair makes more DNA available for sequencing, which in turn improves the sequence data from which the stored digital information is retrieved.

[0004] Recovering the digital information stored in the DNA may include amplifying the pool of stored DNA molecules by polymerase chain reaction (PCR), and sequencing the PCR products using a DNA sequencer to generate sequence data. The sequence data representing the nucleotide base sequence of the individual DNA molecules in the pool is decoded to regenerate the digital information. If too many DNA molecules in the pool are damaged, it may not be possible to obtain sequence data from a sufficient number of DNA molecules, which in turn may make it difficult or impossible to regenerate the digital information. Error correction techniques and storage redundancy can mitigate the effects of some, but not all types of damage.

[0005] Performing enzymatic repair on damaged DNA can significantly increase the number of single DNA molecules that can be successfully amplified by PCR. Amplification of a greater number of different molecules from a pool of DNA allows more digital information to be reconstructed. In some embodiments, after enzymatic repair, the amount of DNA that can be recovered by PCR can be quadrupled. This can make the difference between being able to recover all digital information and being unable to recover all digital information.

[0006] Different enzymes can be used to repair DNA, and every kind of enzyme is suitable for repairing different types of damage. The example of the enzyme that can work on repairing DNA comprises DNA polymerase, DNA ligase and endonuclease. Different reaction conditions such as enzyme concentration, temperature and exposure to the length of the repair mixture also affect the repair efficiency. Under suitable reaction conditions, the damaged DNA is exposed to suitable enzyme to increase the number of different DNA molecules from the pool of the DNA that can be amplified by PCR, and therefore increases the amount of the sequence data that can be used for decoding and regenerating digital information.

[0007] This summary is provided to introduce selected concepts in a simplified form, which are further described in the detailed description below. This summary is neither intended to identify key features or essential features of the claimed subject matter nor to be used to limit the scope of the claimed subject matter. For example, the term "technology" may refer to system(s) and / or method(s) as allowed by the above content and the entire document. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 is a diagram illustrating devices and techniques that can be used to encode digital information from a computer file in DNA and then regenerate the digital information.

[0009] Figure 2 is a flow chart showing an illustrative process for encoding digital information in DNA, storing the DNA, and then decoding sequence data from the DNA to recover the digital information.

[0010] Figure 3 is a flow chart showing an illustrative process for repairing synthetic DNA prior to amplification by PCR and sequencing.

[0011] Figure 4 is a flow chart showing an illustrative process for contacting DNA with a repair mixture if less than a threshold amount of intact DNA is present.

[0012] Figure 5 is a schematic diagram showing a single double-stranded DNA molecule including five separate primer positions that are used to generate three amplification products of different lengths.

[0013] Figure 6 is a bar graph showing that enzymatic repair of DNA molecules doubles the amount of DNA compared to DNA stored under the same conditions without enzymatic repair.

[0014] Figure 7 is a bar graph showing that a four hour incubation with the repair mixture produced higher DNA concentrations than shorter incubation times.

[0015] Figure 8 is a bar graph showing that a four hour incubation with the repair mixture produced higher DNA concentrations than longer incubation times. DETAILED DESCRIPTION

[0016] Polynucleotides such as DNA can be used to store digital information by designing a sequence of nucleotide bases (adenine (A), cytosine (C), guanine (G) and thymine (T)), which encode zeros and ones of digital information. There are various techniques and encoding schemes known to those skilled in the art for using nucleotide bases to represent digital information. See Grass et al. Robust Chemical Preservation of Digital Information on DNA in Silica with Error-correcting Codes, 54 Angew. Chem. Int. Ed. 2552 (2015) and Organick et al. Random access in large-scale DNA data storage, 36: 3 Nat. Biotech. 243 (2018). Advantages of using DNA instead of another storage medium to store digital information include information density and lifespan. The DNA repair technology described in the present disclosure can improve the ability to recover sequence information from damaged DNA that could not be recovered, and reduce the ability to reduce the amount of DNA that must be sequenced in order to be able to regenerate the encoded digital information.

[0017] The disclosure can be used together with any type of double-stranded (ds) polynucleotide, so the reference to " DNA " is illustrative, and is not intended to limit the application to the polynucleotide of a particular type. Ribonucleic acid (RNA), DNA-RNA hybrids, nucleotides including non-natural bases such as dNaM and dTPT3, and the use of nucleotides including less than all four natural bases are also included in the technical scope described in the disclosure. Therefore, " polynucleotide " used in this article can include ds-DNA and ds-RNA with natural base and / or non-natural base. DNA used in this article also includes DNA chains with non-natural base and / or including less than all of natural base.

[0018] Figure 1A schematic representation of using DNA to store digital information and regenerating digital information from DNA is illustrated. Computer file 100 represents any type of file or document that can be accessed by a conventional computer. For example, file 100 can be a video file or a word processing document. File 100 is an example of digital information 102. However, digital information 102 can also include other types of binary sequences, such as sequences that are not encoded in computer file 100.

[0019] Digital information 102 can be converted into a string of nucleotide bases. The number of nucleotide bases required for encoding the entirety of digital information 102 can exceed the length of a single synthetic DNA molecule. Therefore, the nucleotide encoding of digital information 102 is divided into multiple nucleotide sequences 104. There may be hundreds, thousands or millions of nucleotide sequences 104 required to represent a single file 100. At this stage, nucleotide sequence 104 is not a DNA molecule, only the string of characters in the nucleotide base (e.g., a letter sequence of about 80-150 characters long) produces digital information 102 when recombined and correctly decoded. These letter strings provide instructions for establishing a pool of DNA molecules, which will store digital information 102 together. Nucleotide sequence 104 can include subsequences that achieve purposes other than encoding digital information 102, such as, for example, the identification of file 100 and the identification of how each nucleotide sequence in a separate nucleotide sequence 104 should be recombined with each other.

[0020] Once nucleotide sequence 104 is designed, each nucleotide string of these nucleotide strings is provided to oligonucleotide synthesizer 106, and this oligonucleotide synthesizer 106 creates the pool of synthetic DNA 108 with specified sequence.Oligonucleotide synthesizer 106 performs the chemical synthesis of polynucleotide by nucleotide being connected in the sequence specified by nucleotide sequence 104.Utilize current polynucleotide synthesis technology, chain grows along 3 ' to 5 ' direction, and relative to natural biosynthesis, this direction is backward.As chemical process, incorrect interaction may occur, resulting in defective product.The longer the polynucleotide sequence synthesized, the more defects there are, therefore, with regard to current technology, this process is only practical for producing relatively short nucleotide sequence.For DNA molecule with enough quality, current actual limitation is about 100-200 base pairs (bp).Therefore, digital information 102 is divided into a plurality of nucleotide sequences 104, and each nucleotide sequence has the length in the range of about 100 to 200bp. Each nucleotide sequence in nucleotide sequence 104 may be synthesized multiple times by oligonucleotide synthesizer 106 to create multiple synthetic DNA molecules, each of which has the same sequence.

[0021] The synthetic DNA 108 produced by the oligonucleotide synthesizer 106 is a collection of independent DNA molecules having a sequence 102 indicated by a nucleotide sequence 104 and which collectively encodes digital information (including any errors that may be introduced by the synthesis process). In the pool of synthetic DNA 108, there may be redundancy because multiple molecules have the same sequence, as well as diversity due to the inclusion of multiple different nucleotide sequences 104.

[0022] The synthetic DNA 108 may be placed in a storage device 110 until the file 100 is needed to be retrieved. The storage device 110 may be a short-term storage device such as in a buffered aqueous solution. The storage device 110 may be a long-term storage device such as an archival storage device, where the synthetic DNA 108 is kept in a dry or other preserved state for decades, hundreds of years, or even thousands of years. Regardless of the storage technology, the synthetic DNA 108 may be damaged while in the storage device 110.

[0023] DNA can undergo many types of damage, such as hydrolytic damage, oxidative damage, radiation damage, and chemical damage. Hydrolytic DNA damage involves the deamination or complete removal of a single base. AP (apurinic / apyrimidinic) sites are chemically weak and susceptible to single-strand breaks. Hydrolytic damage can be caused by biochemical reactions of various metabolites and an excess of reactive oxygen species. Oxidative DNA damage refers to the oxidation of specific bases. 8-hydroxydeoxyguanosine (8-OHdG) is the most common marker for oxidative DNA damage. 8-OHdG is most commonly formed and enhanced by chemical carcinogens. Ultraviolet light and other types of radiation can damage DNA in the form of DNA strand breaks. This involves the cutting of one or both DNA strands. Ultraviolet-induced damage can also lead to the generation of pyrimidine dimers, in which covalent crosslinking occurs in cytosine and thymine residues. The most common pyrimidine dimers are cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). Chemical DNA damage can be caused by exposure to polycyclic aromatic hydrocarbons (PAHs). PAHs are powerful, ubiquitous atmospheric pollutants that are commonly associated with oil, coal, cigarette smoke, and automobile exhaust. A common marker for DNA damage due to PAHs is benzo(a)pyrene diol epoxide (BPDE). BPDE has been found to be very reactive and is known to covalently bind to proteins, lipids, and guanine residues of DNA to produce BPDE adducts. Damaged DNA may be present in storage device 110, and the type and extent of damage may prevent analysis of the DNA, which in turn prevents recovery of digital information 102.

[0024] Double-strand breaks are difficult to repair because the two parts of the DNA strand can become separated from each other and cannot reconnect. Single-strand breaks are easier to repair because the backbone of the complementary scaffold allows the two strands to anneal to each other and preserve the overall structure of the DNA strand. If the damage results in an abnormally damaged base (e.g., 8-OHdG) or a single-strand break, the technology included in the present disclosure is useful.

[0025] The DNA molecules extracted from the storage device 110 can be repaired using a repair mixture 112 comprising one or more enzymes. The enzymes included in the repair mixture 112 can be polymerases, ligases, endonucleases and / or glycosylases. DNA polymerases are enzymes that synthesize DNA molecules from deoxyribonucleotides (building blocks of DNA). DNA polymerases add nucleotides to the 3' end of a DNA chain, one nucleotide at a time. DNA ligases are specific types of enzymes (ligases) that jointly promote the connection of DNA chains by catalyzing the formation of phosphodiester bonds. Ligase is an enzyme that can catalyze the connection of two macromolecules by forming new chemical bonds. Endonucleases are enzymes that cleave phosphodiester bonds within a polynucleotide chain. Endonucleases play a role in DNA repair. AP endonucleases specifically catalyze the cutting of DNA only at the AP site and thus prepare DNA for subsequent excision, repair synthesis and ligation reactions. Glycosylases are enzymes that hydrolyze glycosyl compounds. DNA glycosylases are a family of enzymes involved in base excision repair. Base excision repair is a mechanism for removing and replacing damaged bases in DNA. DNA glycosylases catalyze the first step in this process, removing the damaged nitrogenous base while leaving the sugar-phosphate backbone intact and creating an AP site.

[0026] DNA removed from storage device 110 can be incubated under a number of different conditions with repair mixture 112. For example, DNA from storage device 110 can be combined with repair mixture 112 at 37°C for a number of hours, such as two, three, four, five, or six hours.

[0027] After contacting the DNA from the storage device 110 with the repair mixture 112, the repaired DNA 114 can be placed in a thermal cycler 116 and amplified by PCR. PCR is a known technique used in molecular biology to exponentially amplify a single copy or several copies of a specific fragment of DNA to generate thousands to millions of copies of the special DNA fragment. Many PCR methods rely on thermal cycling. Thermal cycling exposes the reactants to repeated heating and cooling cycles to allow different temperature-dependent reactions, particularly DNA melting and enzyme-driven DNA replication. PCR uses two main reagents: primers (short single-stranded DNA fragments, referred to as oligonucleotides, which are complementary sequences to the target DNA region) and DNA polymerase. In the first step of PCR, the two chains of the DNA double helix are physically separated at high temperatures in a process known as DNA melting. In the second step, the temperature is lowered, and the primers are bound to the complementary sequences of the DNA. Then, the two DNA chains become templates for DNA polymerase to enzymatically assemble new DNA from free nucleotides (A, C, G and T of DNA). As PCR proceeds, the generated DNA itself is used as a template for replication, initiating a chain reaction in which the original DNA template is exponentially amplified. Thermocycler 116 is a machine that is commonly used to amplify fragments of DNA via PCR. The design of most thermocyclers 116 includes a thermal block with a hole into which a tube containing a reaction mixture can be inserted. Thermocycler 116 then raises and lowers the temperature of the block in discrete, pre-programmed steps to provide thermal cycling.

[0028] PCR amplification increases the number of DNA chains available for analysis by the DNA sequencer 118. However, PCR can introduce errors, so it is possible that the DNA molecules produced by PCR are not all identical copies of the repaired DNA 114. Not all types of DNA sequencing require the use of a thermal cycler 116 and amplification of the repaired DNA 114 by PCR. Therefore, the techniques provided in the present disclosure are equally applicable to implementations that do not use PCR as a pre-sequencing step.

[0029] DNA sequencer 118 detects the sequence of single nucleotides in a DNA sample and generates sequence data 120 representing the sequence. Considering a sample of DNA, such as a PCR product, DNA sequencer 118 determines the order of nucleotide bases. This is then reported as sequence data 120, which can be a text string called "read". The read can be in any suitable format, such as plain text, FASTQ, EMBL or FASTA. There are many different types of DNA sequencers 118 and sequencing technologies. The content of the present disclosure can be used together with any method or technology that can determine the order of nucleotide bases in a chain of DNA.

[0030] The sequencing technology that can be used is synthesis sequencing ( Sequencing by synthesis). Sequencing by synthesis is based on the amplification of DNA on a solid surface using foldback PCR and anchor primers. Adapters are added to the 5' and 3' ends of the fragments. DNA fragments attached to the surface of the flow cell channel are extended and bridged amplified. The fragments become double-stranded, and the double-stranded molecules are denatured. Multiple cycles of solid-phase amplification followed by denaturation can create millions of clusters of approximately 1000 copies of single-stranded DNA molecules of the same template in each channel of the flow cell. Primers, DNA polymerase, and four fluorophore-labeled, reversibly terminated nucleotides are used to perform continuous sequencing. After the nucleotides are incorporated, a laser is used to excite the fluorophores, an image is captured, and the identity of the first base is recorded. The 3' terminus and fluorophore from each incorporated base are removed, and the incorporation, detection, and identification steps are repeated. Synthesis sequencing has a relatively low error rate (e.g., less than 1%) and produces a read length of several hundred base pairs. This length is generally sufficient to read the entire length of a single synthetic polynucleotide.

[0031] Another sequencing technology that can be used is nanopore sequencing. A nanopore is a small hole with a diameter of about one nanometer. Due to the conduction of ions through the nanopore, the immersion of the nanopore in a conductive fluid and the application of an electric potential across the nanopore cause a tiny current. The amount of current flowing through the nanopore is sensitive to the size of the nanopore. When a polynucleotide molecule passes through the nanopore, each nucleotide on the polynucleotide molecule blocks the nanopore to varying degrees. Therefore, when a polynucleotide molecule passes through the nanopore, the change in the current passing through the nanopore represents the reading of the polynucleotide sequence. Nanopore sequencing has a higher error rate than synthetic sequencing (e.g., more than 10%). However, the read length of nanopore sequencing is longer - up to 800,000bp.

[0032] The sequence data 120 output by DNA sequencer 118 can be decoded to create regenerated digital information 122. The decoding of sequence data 120 can be the reverse of the process used to encode nucleotide sequence 104. This encoding / decoding process can include error correction and redundancy, so that even if there is an error such as introduced by oligonucleotide synthesizer 106, damage during storage, PCR amplification or DNA sequencer 118, digital information 102 is correctly regenerated. The error introduced by oligonucleotide synthesis, PCR amplification and DNA sequencing usually has the type and scope that can be solved by error correction and redundancy. In the context of digital information recovery, the damage to synthetic DNA 108 must be considered according to the pool of single DNA molecules. If the sequence data 120 encoded by this damaged DNA molecule can be obtained from other DNA molecules in the pool, the damage to the single DNA molecule can have no influence on the final ability to create regenerated digital information 122. However, extensive damage to a large number of individual DNA molecules in the pool of synthetic DNA 108 during storage 110 may cause the recovered digital information 122 to be inaccurate, or may even prevent recovery of the original digital information 102. Therefore, in some instances, enzymatic repair of the synthetic DNA 108 prior to sequencing may be necessary in order to obtain a sufficiently representative sample of the synthetic DNA 108 to recover the stored digital information 102.

[0033] Illustrative Process

[0034] For ease of understanding, the operations discussed in this disclosure are depicted as separate operations as independent boxes. However, these separately depicted operations should not be interpreted as a necessary order in which they are relied upon for execution. The order in which the process is described is not intended to be interpreted as a limitation, and any number of the process boxes may be combined in any order to implement the process or an alternative process. In addition, it is also possible that one or more of the provided operations are modified or omitted. Figure 2-4 Can be Figure 1 The systems and devices shown may be implemented in whole or in part.

[0035] Figure 2 A process 200 for repairing stored DNA encoding digital information is shown.

[0036] At 202, digital information is encoded as a sequence of nucleotides. The digital information may represent all or part of a computer file or an executable computer program. The encoding may use any encoding technique known to those skilled in the art for translating binary information into a code comprising nucleotide bases. The encoding scheme may include error correction, such as, for example, Hamming code, Reed-Solomon code, multidimensional parity check, Bose-Chaudhuri-Hocquenghem code, or any other suitable error correction code.

[0037] At 204, a synthetic DNA molecule is synthesized. The synthetic DNA molecule includes at least a portion of a sequence of nucleotides encoding digital information. The synthetic DNA molecule may also include nucleotides encoding other information. The synthetic DNA molecule may be synthesized by an oligonucleotide synthesizer such as Figure 1 The length of the synthetic DNA molecule can be between about 80-250 bp, between about 100-180 bp, or between about 120-160 bp.

[0038] At 206, the synthetic DNA molecules are stored. The synthetic DNA molecules can be stored using any technique for storing DNA. For example, the synthetic DNA molecules can be stored in a buffered aqueous solution (e.g., Tris-EDTA) and refrigerated at about 4°C. Additionally, the synthetic DNA molecules can be frozen at -20°C or stored as a precipitate in ethanol at -80°C. The DNA can also be stored in liquid nitrogen at -196°C. The synthetic DNA molecules can be stored at room temperature, such as by drying the DNA and storing it on paper cards (e.g., card) or another substrate.

[0039] At 208, the synthetic DNA molecules are contacted with a repair mixture. The repair mixture may be Figure 1 The repair mixture 112 introduced in the present disclosure. The repair mixture includes at least one enzyme and may include any or all of a DNA polymerase, a DNA ligase, or an endonuclease. For all enzymes described in the present disclosure, enzymes derived from natural sources or enzymes obtained from recombinant sources may be used.

[0040] A suitable DNA polymerase that can be used is Bst DNA polymerase. Bst polymerase is derived from the Gram-positive bacterium Bacillus stearothermophilus. Bst polymerase has helicase-like activity, enabling it to unwind DNA strands.

[0041] A suitable DNA ligase that can be used is Taq DNA ligase. Taq DNA ligase is a thermostable ligase that catalyzes the formation of a phosphodiester bond between the 5'-phosphate and 3'-hydroxyl groups of two adjacent DNA strands. The enzyme is derived from the thermophilic bacterium Thermus aquaticus.

[0042] One suitable endonuclease that can be included in the repair mixture is T4 endonuclease V, originally isolated from Enterobacteriaceae phage T4. The enzyme has both DNA glycosylase and APlyase activities. T4 endonuclease V recognizes cis-syn-cyclobutane pyrimidine dimers caused by ultraviolet irradiation. The enzyme cleaves the glycosyl bond at the 5'-end of the pyrimidine dimer, and the endonucleolytic activity cleaves the phosphodiester bond at the AP site.

[0043] A suitable endonuclease that can be used for DNA repair is endonuclease IV. Endonuclease IV can act on a variety of oxidative damage in DNA. The enzyme is an apurinic / apyrimidinic (AP) endonuclease that hydrolyzes intact AP sites in DNA. The AP site is cleaved to the lesion at the first phosphodiester bond that is 5', leaving a hydroxyl at the 3' terminus and a deoxyribose 5'-phosphate at the 5' terminus. The enzyme also has 3'-diesterase activity and can release phosphoglyceraldehyde, intact deoxyribose 5-phosphate and phosphate from the 3'-end of the DNA.

[0044] Another suitable endonuclease that can be included in the repair mixture is endonuclease VIII. This endonuclease comes from the E.coli that serves as N-glycosylase and AP-lyase. N-glycosylase activity releases damaged pyrimidine from double-stranded DNA, generates apurine (AP site). AP-lyase activity cracks 3' and 5' to the AP site, leaving 5'-phosphate and 3'-phosphate. The damaged bases identified and removed by endonuclease VIII include urea, 5,6-dihydroxy thymine, thymine diol, 5-hydroxy-5-methylhydantoin, uracil diol, 6-hydroxy-5,6-dihydrothymine and methyl tartaryl urea (1,2). When endonuclease VIII is similar to endonuclease III, endonuclease VIII has β and δ lyase activity, and endonuclease III only has β lyase activity.

[0045] Contacting the synthetic DNA molecule with the repair mixture can be performed under a variety of reaction conditions. The synthetic DNA molecule can be incubated using a reaction mixture at a temperature of 20-60°C, 30-50°C, 35-45°C or about 37°C. The duration of incubation can be 2-8 hours, 3-5 hours or about 4 hours. It is worth noting that before the synthetic DNA is contacted with the repair mixture, the synthetic DNA can be maintained at about room temperature or a colder temperature. Therefore, the previous steps of process 200 can be performed at a temperature less than 25°C, less than 22°C, less than 19°C, less than 16°C or lower. As used herein, "approximately" or "about" represents a range of ±10% of the value.

[0046] At 210, after contacting with the repair mixture, the synthetic DNA is sequenced to generate sequence data. Sequencing can be performed by any suitable technique for DNA sequencing. For example, sequencing can be performed by Figure 1 The process is performed by the DNA sequencer 118 introduced in the invention.

[0047] At 212, the sequence data obtained at 210 is decoded to obtain the regenerated digital information. If the synthetic DNA has been adequately repaired, and any other errors introduced in the process have been mitigated, the regenerated digital information will contain a binary sequence that is identical or nearly identical to the digital information originally encoded in the nucleotides.

[0048] Figure 3 A process 300 is shown for presequencing processing of DNA including enzymatic repair.

[0049] At 302, the synthetic DNA is contacted with a repair mixture. The repair mixture may be Figure 1 The repair mixture 112 introduced in the invention. The repair mixture comprises one or more enzymes suitable for repairing DNA. In an embodiment, the repair mixture comprises at least one of Bst DNA polymerase, Taq DNA ligase, endonuclease IV, T4 endonuclease V, or endonuclease VIII. In an embodiment, the repair mixture comprises Bst DNA polymerase, Taq DNA ligase, endonuclease IV, T4 endonuclease V, and endonuclease VIII. The repair mixture can also be formulated so that it only includes a single enzyme. For example, the repair mixture can only include the enzyme T4 endonuclease V or endonuclease IV. The repair mixture formulated with a single enzyme can include other non-enzyme components, such as water, buffer, dNTPs, and oxidized nicotinamide adenine dinucleotide (NAD + ).

[0050] The synthetic DNA can be a DNA synthesized by an oligonucleotide synthesizer such as oligonucleotide synthesizer 106. The synthetic DNA has a sequence that is predetermined at least in part by the encoding of digital information. The length of the synthetic DNA can be approximately between 50-200 bp, 80-180 bp, or 120-160 bp. The contacting of the synthetic DNA with the repair mixture can be performed at a temperature of about 37° C. for about four hours.

[0051] At 304, the synthetic DNA is amplified by PCR. PCR can be performed by, for example, Figure 1 In some embodiments, the invention relates to a method for repairing a synthetic DNA by contacting with a repair mixture to repair the synthetic DNA. The number of complete chains of the DNA that can be amplified by the primers used for PCR is increased. Compared with not using the repair mixture to amplify the synthetic DNA, this causes an increase in the number of different DNA chains from the pool of DNA. Therefore, the amplified product after repair provides a better representation of the multiple DNA sequences stored in the pool of DNA. Compared with the PCR amplification of unrepaired synthetic DNA under the same conditions, the growth of the number of different DNA chains amplified due to the use of the repair mixture to prepare DNA can be about 1.5 times, 1.8 times, 2.0 times, 4.0 times or more growth. Therefore, in some implementations, contacting the synthetic DNA with the repair mixture at 302 can double the amount of the DNA produced by PCR at 304.

[0052] At 306, the synthesized DNA after PCR amplification is sequenced. Sequencing can be performed by any suitable technique to read the nucleotide sequence of a DNA molecule, such as in Figure 1 The DNA sequencer 118 introduced in the invention. Due to the use of repair mixture, the growth of the number of complete DNA chains provides more different molecules of DNA to be sequenced. A larger number of different DNA molecules allow sequencing to capture more multiple DNA stored in the DNA pool sequence, and under non-strict sequencing conditions, sequence data digital information can be allowed to be decoded. A way in which sequencing conditions can be relaxed is by reducing coverage or reading depth. The coverage (or depth) of DNA sequencing is the number of unique reads including a given nucleotide in the reconstructed sequence.

[0053] Figure 4 A process 400 for recovering digital information from a damaged DNA strand is shown. The goal of recovering digital information from DNA used for data storage is to be able to reconstruct the original digital information without errors. Figure 1 This is illustrated by a sequence of regenerated digital information 122 that is identical to digital information 102. Damage to the DNA strands during storage can be considered as "noise" in the communication channel. DNA repair is a technique used to reduce noise.

[0054] At 402, it is determined that the number of intact DNA chains in the sample of synthetic DNA is less than a threshold level. The number of intact chains of DNA in the sample can be measured by quantitative PCR (qPCR). qPCR uses the linearity of DNA amplification to determine the number of DNA in the sample. By using fluorescent reporter molecules in the reaction, it is possible to measure DNA generation in real time in a qPCR experiment as the PCR reaction proceeds. The number of PCR cycles at which the fluorescence exceeds the detection threshold (e.g., 0.2 fluorescence units) and becomes measurable is called a quantitative cycle (Cq). A starting sample that includes a large amount of amplifiable DNA (i.e., intact DNA chains with primer binding sites) reaches the threshold using fewer PCR cycles than a sample with a smaller amount of DNA. For a sequence with the best PCR efficiency (100%), each PCR cycle doubles the amount of DNA, so an increase in the Cq value of 1 is equivalent to a 50% reduction in the initial amount of DNA detected. Therefore, the Cq value is inversely proportional to the amount of target DNA in the sample.

[0055] The threshold level can be based on the amount of DNA required for successful recovery of digital information. The amount of DNA is not directly related to the ability to recover digital information. A large number of only a few different DNA chains can provide a reasonable amount of DNA, but the sequence encoded by the DNA chain will not include all of the data required to regenerate the digital information. However, assuming that the damage caused during storage is roughly randomly distributed throughout the pool of DNA molecules, calculations can be performed to identify the amount of DNA that may contain a sufficiently diverse single DNA molecule.

[0056] Threshold level can also be based on the change of Cq value for synthetic DNA before and after storage.After the synthesis of synthetic DNA, the sample in the pool can be adopted, and the quantity of DNA is measured using qPCR.This provides a starting value for the amount of DNA before storage.After storage, the Cq value is measured again.As mentioned above, the Cq value reduces a roughly indicating halving of available DNA.If the difference between Cq values ​​is greater than the threshold number (e.g., 1.0, 2.0, 3.0).The threshold number can be experimentally determined based on the experiment of recovering digital information from other pools of DNA.The technology for encoding digital information in synthetic DNA and the redundancy level in the synthesis of synthetic DNA molecules can affect the threshold.

[0057] At 404, the synthetic DNA is contacted with a repair mixture. The repair mixture includes at least one of a DNA polymerase, a DNA ligase, or an endonuclease. The repair mixture may be contacted with Figure 1 Alternatively, if it is determined that there are sufficient numbers of intact DNA chains to proceed, the synthetic DNA can be sequenced without first using a repair mixture.

[0058] At 406, the synthetic DNA is sequenced to generate sequence data. The synthetic DNA may be sequenced by Figure 1 The DNA sequencer 118 described in the present invention is used for sequencing. Depending on the amount of DNA and the sequencing technology used, sequencing may be preceded by PCR amplification of the repaired DNA.

[0059] At 408, the sequence data is decoded to obtain regenerated digital information.

[0060] Example

[0061] Figure 5 A schematic diagram of synthesizing a dsDNA chain 500 and five different primers bound to the DNA chain 500 is shown. The DNA chain 500 has a length of 113 bp (SEQ ID: 1). This DNA chain 500 is used in the following examples.

[0062] The first forward primer (Primer1F 502 (SEQ ID: 2)) binds to one end of the DNA chain 500. When paired with the first reverse primer (Primer1R 504 (SEQ ID: 3)) that binds to the other end of the DNA chain 500, the amplification product is the entire 113 bp length of the DNA chain 500. The second forward primer (Primer2F 506 (SEQ ID: 4)) binds to the DNA chain 500 that partially overlaps with the binding site of Primer1F 502. The PCR amplification product using Primer2F 506 as the forward primer and the second reverse primer (Primer2R 508 (SEQ ID: 5)) as the reverse primer is an 83 bp long fragment from the middle of the DNA chain 500. The third primer (Primer3R 508 (SEQ ID: 6)) located near the middle of the DNA strand 500 creates an amplification product that is 53 bp long when used with the second forward primer (Primer2F 506).

[0063] These three different length sequences that can be created from DNA strand 500 are used to test the effect of strand length on DNA repair and recovery.

[0064] Effects of chain length and enzymatic repair on DNA recovery

[0065] Figure 6 A bar graph 600 is shown comparing the amount of intact DNA in samples with and without enzymatic repair. Three DNA samples of different lengths, 53 bp, 83 bp, and 113 bp, were prepared using the above primers. Figure 5DNA samples were exposed to high heat and humidity to simulate aging. In this example, the DNA samples were stored in an oven at 70°C and 50% relative humidity (RH) for two hours. This simulates approximately 15 years of storage at room temperature and 20% RH. Exposure to high heat and humidity is believed to cause primarily hydrolytic damage to the DNA samples. Without being bound by theory, it is believed that damage to the DNA samples includes breaks in the backbone of the DNA strands.

[0066] The initial concentration of the DNA samples, shown as 100% on the vertical axis of bar graph 600, was determined by measuring the DNA concentration for each sample before exposure to high heat and humidity. In this example, quantitative PCR (qPCR) was used to measure the amount of DNA in the samples before and after exposure to high heat and humidity. Real-time PCR cycler to perform qPCR.

[0067] Without enzymatic repair, more than half of the available DNA is lost during exposure to high heat and humidity. The percentage of DNA loss increases with the length of the DNA sample. For a 53bp DNA strand, about 55% of the original DNA is lost, and for a 113bp DNA strand, about 65% of the original DNA is lost.

[0068] However, using enzymatic repair, the amount of intact DNA that can be recovered is approximately twice that of the unrepaired sample. For the 53bp DNA strand and the 83bp DNA strand, approximately 85% of the original DNA amount is recovered after enzymatic repair using the repair mixture. For the longer 113bp DNA sample, more than 70% of the original DNA amount is recovered.

[0069] In this example, the repair mixture was available from New England BioLabs Inc. (registration #M0309S). Repair Mixture( Repair Mix). Repair Mix includes TaqDNA ligase, Endonuclease IV, Bst DNA polymerase, Formamidopyrimidine DNA glycosylase (Fpg), Uracil-DNA glycosylase (UDG), T4 PDG (T4 Endonuclease V), and Endonuclease VIII. Repair Mix, dNTPs, and NAD+ were incubated in a buffer solution at 37°C with shaking for four hours. The content of the Repair Mix is ​​known, but the concentration of the enzyme is unknown.

[0070] Comparison of incubation time

[0071] Figure 7 and Figure 8 The bar graphs 700 and 800 show the effect of repair time on the amount of DNA recovered from a damaged DNA sample. Figure 7 and Figure 8 The samples analyzed in Figure 5 The full-length 113 bp DNA chain described in the above was 500. The repaired sample was used under the above conditions Repair Mix is ​​incubated. The amount of DNA is used The qPCR was used to measure

[0072] exist Figure 7 The sample shown in bar graph 700 was exposed to a temperature of 70°C and 50% RH for three hours. The column labeled "Reference" represents the amount of DNA in the sample before exposure to high heat and humidity. In the absence of repair, less than 40% of the original amount of DNA was recovered. As the incubation time with the repair mixture increased, the amount of DNA recovered gradually increased. After four hours of incubation with the repair mixture, the highest recovery of approximately 70% was obtained.

[0073] Figure 8 The sample values ​​shown in bar graph 800 are used to generate Figure 7 The data shown were generated under the same conditions, except that the exposure to 70°C temperature and 50% RH was for four hours. After this period of exposure to high heat and humidity, only about 20% of the unrepaired DNA sample remained. Interestingly, increasing the length of incubation with the repair mixture beyond four hours reduced the amount of DNA recovered. In this set of samples, the highest amount of DNA recovery of about 45% of the original amount was obtained after four hours of incubation with the repair mixture. Figure 7 and Figure 8 The difference in the amount of DNA recovered between the four hours of incubation may be due to Figure 8 Increased exposure of samples to high heat and humidity.

[0074] Comparison of repair mixtures

[0075] Table 1 below compares the ability of different enzyme combinations to effectively repair damaged DNA. The enzymes tested were Bst DNA polymerase, T4 endonuclease V, endonuclease IV, endonuclease VIII, DNA ligase, and uracil-DNA glycosylase (UDG). All enzymes were obtained from New England BioLabs Inc. and prepared according to the manufacturer's specifications including standard dilutions in the provided buffer. DNA samples were from Figure 5 The DNA strand of 500 has a 113 bp full-length sequence. Damage from long-term storage was simulated by exposing the DNA samples in solution to 70°C and 50% RH for 24 hours.

[0076] In addition to the premix solution, each sample was prepared by mixing 5 ng of DNA with 50 μL of ultrapure water. The premix solution included 5 μL of 5% dHO available from New England BioLabs, Inc. (registration # B9004S). Reaction buffer, 0.5 μL of 10 mM dNTP mixture and 0.5 μL of 50 mM NAD+. As used herein, "ultrapure water" refers to water having a purity such that the resistivity is 18.2 MΩ·cm at 25°C or "Type 1" water as specified in ISO (International Organization for Standards) 3696. One source of ultrapure water is Milli-Q TM water.

[0077] The amount of DNA available after repair was determined using Roche qPCR was used to measure

[0078] The "undamaged" sample is a sample of DNA strands that were not subjected to high heat and high humidity. As expected, this sample had a lower Cq value than any other sample. The "no repair" sample is a sample of DNA that was not incubated with a repair mixture. The Cq value of this sample was 31.11. Therefore, a Cq value below 31.11 indicates a benefit from enzymatic repair. Values ​​above 31.11 indicate that the repair mixture did not produce any benefit under these conditions.

[0079] The "control" sample was a sample of DNA incubated with the premix solution without any enzyme. The Cq value of 27.59 for the control sample was lower than the Cq values ​​for many of the reaction mixtures that included enzymes. Without being bound by theory, it is believed that the success of the premix solution in repairing damage is an artifact when dealing with very low DNA concentrations. Similar benefits were not expected at higher DNA concentrations.

[0080]

[0081]

[0082] Table 1 Comparison of the ability of various enzyme combinations to repair damaged DNA

[0083] Surprisingly, the repair mixtures 17 and 18 that only comprise a single endonuclease, T4 endonuclease V and endonuclease IV, respectively, perform better than the repair mixtures with multiple enzymes. Without being bound by theory, it is believed that multiple enzymes can interfere with each other and reduce the ability to repair damaged DNA. Although the repair mixture comprising multiple enzymes can be used to repair the damage of the DNA chains that have suffered many different types of damage, if the DNA chains are damaged in the same manner, the mixture of enzymes can reduce the repair efficacy.

[0084] Table 2 compares the repair mixtures with single endonucleases. Repair Mix's repair efficacy. Two DNA strands of different lengths were tested: Figure 5 The sample preparation and testing were performed in the same manner as described above for the results shown in Table 1.

[0085]

[0086] Table 2 Comparison of repair mixtures containing individual endonucleases with commercial DNA repair mixtures

[0087] These comparisons showed that the length of the DNA strands had a small effect on the ability of the DNA to be repaired. The combination of Taq DNA ligase, endonuclease IV, Bst DNA polymerase, formamidopyrimidine DNA glycosylase (Fpg), uracil-DNA glycosylase, T4 endonuclease V, and endonuclease VIII in the Repair Mix repaired damage to these DNA strands more efficiently than any of the three endonucleases alone. The individual concentrations of the various enzymes in the Repair Mix are unknown, so the discrepancy may be due in part to concentration variations rather than the combination of enzymes used.

[0088] Table 3 compares the effects of enzyme concentration and incubation time on repair efficacy. The concentrations of dNTPs and NAD+ in the Repair Mix and buffer were used at the standard concentrations recommended by the manufacturer (indicated by "x") and at twice the recommended concentrations (indicated by "xx"). Figure 5 The DNA samples in solution were exposed to 70°C and 50% RH for 24 hours to simulate damage from long-term storage. The damaged DNA was incubated with a repair mixture for three, four or five hours.

[0089] The "undamaged" sample is a sample of DNA strands that have not been subjected to high heat and humidity. However, this sample may have a certain degree of damage from short-term storage before testing. The "no repair" sample is a DNA sample that has not been incubated with a repair mixture. The Cq value of this sample is 7.91. Therefore, in this comparison, a Cq value below 7.91 indicates a benefit from enzymatic repair.

[0090]

[0091]

[0092] Table 3 Comparison of enzyme concentrations and incubation times.

[0093] All samples in this comparison showed improvements over the "no repair" sample. Incubation for four hours produced lower Cq values ​​than for three hours or for five hours, thus producing higher DNA quantities. Doubling the concentration of the Repair Mix to twice the level specified in the manufacturer's recommendations increased the amount of DNA that could be repaired. Doubling the amount of dNTPs also improved repair efficiency. However, doubling the amount of NAD+ had the opposite effect.

[0094] The best result, sample number 7 with a Cq value of 6.04, was obtained using twice This was achieved with the standard amount of Repair Mix and twice the amount of dNTPs. This is a lower Cq value than the Cq value of the undamaged sample, indicating that a certain degree of damage can exist even without exposure to high heat and humidity, and that the damage is repaired by the Repair Mix.

[0095] Illustrative Embodiments

[0096] The following clauses describe a plurality of possible embodiments for realizing the features described in this disclosure. The various embodiments described in this article are not restrictive, and each feature from any given embodiment is not required to exist in other embodiments. Unless the context clearly indicates otherwise, any two or more embodiments can be combined together. As used herein in this document, "or" means and / or. For example, "A or B" means A without B, B without A, or A and B. As used herein, "comprising" means including all listed features and may include the addition of other features not listed. "Substantially consisting of..." means including the listed features and those additional features that will not substantially affect the basic characteristics and novel characteristics of the listed features. "Consisting of..." only means the listed features and excludes any features not listed.

[0097] Item 1. A method of storing digital information in deoxyribonucleic acid (DNA), comprising: encoding the digital information as a sequence of nucleotides; synthesizing a synthetic DNA molecule having the sequence of nucleotides; storing the synthetic DNA molecule; contacting the synthetic DNA molecule with a repair mixture comprising at least one of: a DNA polymerase, a DNA ligase, or an endonuclease; after contacting with the repair mixture, sequencing the synthetic DNA molecule to generate sequence data; and decoding the sequence data to obtain regenerated digital information.

[0098] Clause 2. The method of clause 1, wherein encoding the digital information uses a coding scheme that includes error correction.

[0099] Clause 3. The method according to clause 1 or clause 2, wherein synthesizing the synthetic DNA molecule is performed by an oligonucleotide synthesizer, and the DNA molecule is between about 100-180 base pairs (bp) long.

[0100] Clause 4. The method according to any one of clauses 1-3, wherein contacting the synthetic DNA molecule with the repair mixture is performed at about 37°C for about 4 hours.

[0101] Clause 5. The method of any one of Clauses 1-4, wherein, subsequent to synthesizing the synthetic DNA molecule, the synthetic DNA molecule is maintained at a temperature of no greater than about 25°C until contacting the synthetic DNA molecule with the repair mixture.

[0102] Clause 6. The method according to any one of clauses 1 to 5, wherein the repair mixture comprises Bst DNA polymerase.

[0103] Clause 7. The method according to any one of clauses 1 to 6, wherein the repair mixture comprises Taq DNA ligase.

[0104] Clause 8. The method of any one of clauses 1 to 7, wherein the repair mixture comprises at least one of: T4 Endonuclease V, Endonuclease IV, or Endonuclease VIII.

[0105] Clause 9. A method of repairing synthetic DNA encoding digital information, comprising: contacting the synthetic DNA with a repair mixture comprising at least one of: a DNA polymerase, a DNA ligase, or an endonuclease; and amplifying the synthetic DNA by polymerase chain reaction (PCR), wherein PCR amplification of the synthetic DNA after contact with the repair mixture reduces the Cq cycle number by at least 1.5 compared to PCR amplification of the synthetic DNA not contacted with the repair mixture.

[0106] Clause 10. The method according to Clause 9, wherein the synthetic DNA has a length of about 100-180 bp and is synthesized by an oligonucleotide synthesizer according to a predetermined code of digital information.

[0107] Clause 11. The method according to clause 9 or 10, wherein the contacting of the synthetic DNA with the repair mixture is performed at about 37°C for about 4 hours.

[0108] Clause 12. The method according to any one of clauses 9 to 11, wherein the repair mixture comprises at least one of the following: Bst DNA polymerase, Taq DNA ligase, Endonuclease IV, T4 Endonuclease V or Endonuclease VIII.

[0109] Clause 13. The method according to any one of clauses 9 to 12, wherein the repair mixture comprises Bst DNA polymerase, Taq DNA ligase, endonuclease IV, T4 endonuclease V and endonuclease VIII.

[0110] Clause 14. The method according to any one of clauses 9 to 13, wherein the repair mixture consists essentially of T4 Endonuclease V.

[0111] Clause 15. The method according to any one of clauses 9 to 13, wherein the repair mixture consists essentially of Endonuclease IV.

[0112] Clause 16. The method of any one of clauses 9 to 15, wherein PCR amplification of the synthetic DNA following contact with the repair mixture reduces the Cq cycle number by at least 2.0 compared to PCR amplification of synthetic DNA not contacted with the repair mixture.

[0113] Clause 17. The method according to any one of clauses 9 to 16, further comprising sequencing the synthetic DNA after PCR amplification.

[0114] Clause 18. A method for recovering digital information stored in synthetic DNA comprises: determining that the number of intact DNA strands in the synthetic DNA is less than a threshold level; contacting the synthetic DNA with a repair mixture comprising at least one of: a DNA polymerase, a DNA ligase, or an endonuclease; and sequencing the synthetic DNA to generate sequence data.

[0115] Clause 19. The method according to clause 18, wherein the threshold level is based on the difference between the Cq value of the synthesized DNA before storage and the Cq value of the synthesized DNA after storage.

[0116] Clause 20. The method of clause 18 or 19, further comprising decoding the sequence data to obtain regenerated digital information.

[0117] in conclusion

[0118] Although the subject matter has been described in language specific to structural features and / or methodological acts, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts are disclosed as example forms of implementing the claims.

[0119] The terms "a," "an," "the," and similar referents used in the context of describing the present invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "based on," "according to," and similar referents should be construed to mean "based at least in part on," including "based in part on," and "based entirely on," unless otherwise indicated herein or clearly contradicted by context.

[0120] Certain embodiments are described herein, including the best mode known to the inventor for carrying out the invention. Of course, variations of these described embodiments will become apparent to those of ordinary skill in the art after reading the foregoing description. The skilled person will know how to appropriately adopt such variations, and the embodiments disclosed herein may be practiced differently than specifically described. Therefore, all modifications and equivalents of the subject matter recited in the appended claims are included within the scope of this disclosure. Moreover, unless otherwise indicated herein or clearly contradictory to the context, the present invention encompasses any combination of the above elements in all possible variations thereof.

Claims

1. A method for increasing the amount of synthetic double-stranded DNA that can be amplified by PCR, the synthetic double-stranded DNA comprising natural base-encoded digital information, the method comprising: confirming that the amount of DNA amplifiable by PCR in the synthetic double-stranded DNA is less than a threshold level, wherein the threshold level is based on a change in the amount of DNA amplifiable by PCR before and after storage measured by quantitative PCR; contacting the synthetic double-stranded DNA with a repair mixture comprising an endonuclease; and The synthetic double-stranded DNA was amplified by polymerase chain reaction (PCR).

2. The method according to claim 1, wherein the synthetic double-stranded DNA has a length of 100-180 bp and is synthesized by an oligonucleotide synthesizer according to a predetermined code of the digital information.

3. The method of claim 2, wherein the predetermined encoding of the digital information uses a coding scheme that includes error correction.

4. The method of claim 1, wherein the repair mixture comprises at least one of the following: Bst DNA polymerase, Taq DNA ligase, formamidopyrimidine DNA glycosylase (Fpg), or uracil-DNA glycosylase (UDG).

5. The method according to claim 4, wherein the endonuclease is selected from the group consisting of endonuclease IV, T4 endonuclease V, and endonuclease VIII.

6. The method of claim 1, wherein the repair mixture comprises Bst DNA polymerase, Taq DNA ligase, endonuclease IV, T4 endonuclease V, endonuclease VIII, formamidopyrimidine DNA glycosylase (Fpg), and uracil-DNA glycosylase (UDG).

7. The method of claim 1, further comprising sequencing the synthetic double-stranded DNA after PCR amplification.

8. The method according to claim 7, further comprising decoding the sequence of the synthetic double-stranded DNA obtained by sequencing to obtain digital information.

9. The method according to claim 1, wherein the repair mixture further comprises a DNA polymerase, and wherein the DNA polymerase is Bst DNA polymerase.

10. The method according to claim 1, wherein the repair mixture further comprises DNA ligase, and wherein the DNA ligase is Taq DNA ligase.

11. The method of claim 1, wherein the endonuclease is endonuclease IV, T4 endonuclease V, or endonuclease VIII.

12. The method of claim 11, wherein the repair mixture comprises a plurality of endonucleases, and wherein the plurality of endonucleases comprises endonuclease IV, T4 endonuclease V, and endonuclease VIII.

13. The method of claim 1, wherein the repair mixture further comprises a glycosylase, and wherein the glycosylase is formamidopyrimidine DNA glycosylase (Fpg) or uracil-DNA glycosylase (UDG).

14. The method of claim 13, wherein the repair mixture further comprises a plurality of glycosylases, and wherein the plurality of glycosylases comprises formamidopyrimidine DNA glycosylase (Fpg) and uracil-DNA glycosylase (UDG).

15. The method of claim 1, wherein the synthetic double-stranded DNA is contacted with the repair mixture at 37°C for 4 hours.

16. The method according to claim 1, wherein: The change in the amount of DNA amplifiable by PCR is determined by comparing a first amount of DNA amplifiable by PCR represented by a first Cq value measured before storage and a second amount of DNA amplifiable by PCR represented by a second Cq value measured after storage.

17. The method of claim 16, wherein the threshold level is a numerical difference between the first Cq value and the second Cq value, or the threshold level is based on an amount of DNA required to successfully recover the digital information.

18. The method of claim 1, wherein the repair mixture comprises at least one of a DNA polymerase, a DNA ligase, or a glycosylase, and an endonuclease.

19. The method of claim 18, wherein the repair mixture comprises at least two of a DNA polymerase, a DNA ligase or a glycosylase, and an endonuclease.

20. The method of claim 19, wherein the repair mixture comprises an endonuclease, a DNA polymerase, a DNA ligase, and a glycosylase.

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