A molecular barcode system facilitating the discrimination of biological samples and a method for obtaining said molecular barcode system

Abstract

The invention relates to a molecular barcode system that facilitates the identification of biological samples in order to eliminate potential mixing risks in analysis centers and to resolve problems arising from manual labeling, and to a method for obtaining this molecular barcode system.

Description

[0001] A MOLECULAR BARCODE SYSTEM FACILITATING THE DISCRIMINATION OF BIOLOGICAL SAMPLES AND A METHOD FOR OBTAINING SAID MOLECULAR BARCODE SYSTEM

[0002] TECHNICAL FIELD

[0003] The invention relates to a molecular barcode system that facilitates the identification of biological samples in order to eliminate potential mixing risks in analysis centers and to resolve problems arising from manual labeling, and to a method for obtaining this molecular barcode system.

[0004] PRIOR ART

[0005] Deoxyribonucleic acid, or DNA for short, is a nucleic acid that carries the genetic instructions necessary for the vital functions and biological development of all organisms and some viruses. The primary role of DNA is the long-term storage of information. Since it contains the information necessary for the construction of other components of the cell such as proteins and RNA, DNA is likened to a mold, template, or recipe.

[0006] Nucleobases (or nucleotide bases) are nitrogenous chemical molecules that are bound to the sugars in RNA and DNA. These include adenine, guanine, cytosine, thymine (found only in DNA), and uracil (found only in RNA). They are abbreviated as A, G, C, T, and U, respectively. In genetics, they are commonly referred to as bases.

[0007] Biological samples taken from individuals in the relevant technical field are archived in analysis centers for testing. After storing these biological samples in suitable sterile containers, labeling is performed and the samples are stored specifically for each individual. Another important point to be considered here is labeling. Even the slightest error during labeling can lead to very serious problems. Some of these technical problems include the label being unreadable, the label fading over time, incorrect labeling, and the label falling off.

[0008] The labeling system on the sample taken from individuals is not always carried out in a digital environment but is sometimes handwritten by healthcare personnel. During this handwriting process, any incorrectly written letter or number will make it difficult to identify the sample. Additionally, when problems such as the label detaching or twisting occur during transportation, the labels become difficult to read. A sample with a label that is difficult to read cannot be tested, and even if a test is conducted, it will not yield an accurate result since it will be unclear which patient it belongs to.

[0009] Another technical problem is the erasure of the label. Similarly, the labeling on the sample, whether within the hospital or sent to another testing laboratory, must be proper and legible. The code written on the label is erased from the sample due to external conditions such as temperature and water. A sample with an erased code on its label cannot be tested, and even if a test is conducted, it will not yield an accurate result since it will be unclear which patient it belongs to.

[0010] Along with the sample taken from individuals, healthcare personnel must be even more careful. In hospitals, when sample collection rooms are crowded, it becomes confusing to determine which sample belongs to which individual. If this confusion goes unnoticed and incorrect labeling is performed, it will lead to irreversible consequences. The sample of person A is labeled with the label of person B and sent for testing. Since the test results do not belong to person A, the application of incorrect treatment causes serious problems.

[0011] Another technical problem is the label falling into the sample. A non-sterile label that accidentally falls into the sterile sample container will disrupt the chemistry of the sample. This disrupted sample chemistry does not yield accurate results.

[0012] Patent application number US2021010063 relates to barcoded molecular standards. The said patent covers the purpose of preventing errors that may occur during process steps such as sample transportation, transfer of samples between multiple containers, library preparation, PCR amplification, sequencing runs, and the reporting of results.

[0013] Patent application number US2011202280 relates to the field of DNA sequencing, including systems and methods for detecting and correcting errors or ambiguities encountered in or associated with the sequencing of DNA samples.

[0014] As a result, it has become a necessity to introduce innovations in order to eliminate the above-mentioned technical problems.

[0015] BRIEF DESCRIPTION OF THE INVENTION

[0016] In the relevant technical field, it has been observed that in centers where biological samples are analyzed or particularly in cases where transportation from one place to another is required, the information belonging to the sample may be lost. Especially in the case of samples to be transported over long distances, if the information specific to the sample is lost, it may be very difficult to obtain the same samples again.

[0017] In the relevant technical field, information related to biological samples is mostly provided through manually handwritten labels or packaging materials on tubes. However, these labels and packaging materials may be removable, tearable, prone to the creation of incorrect information, and handwritten texts may be illegible. Furthermore, since biological samples are samples that can be stored for long periods, the fading or illegibility of the writings over time may also occur.

[0018] In order to eliminate all the above-mentioned technical drawbacks, the present inventors propose a barcoding method for the relevant technical field, which facilitates the differentiation of biological samples from other samples and enables the specific storage of information related to the sample.

[0019] The barcoding method subject to the invention provides a method in which technical problems arising from these applications do not occur, since it does not include arrangements for placing information manually or with a packaging material on the tube.

[0020] The invention also relates, in another aspect, to a barcoding system that enables the implementation of the said barcoding method.

[0021] DETAILED DESCRIPTION OF THE INVENTION

[0022] In this detailed description, the invention relates to a method that enables the specific differentiation of biological samples from other samples in analysis centers or during transportation and storage processes, and to a molecular barcode system that enables the implementation of this method, and is explained with examples that are solely intended to provide a better understanding of the subject and do not have any limiting effect.

[0023] The molecular barcode system subject to the present invention is a data system that enables the matching of personal information with a DNA-based encryption system. By creating a unique sequence list for each individual, it will facilitate the differentiation of a person’s samples from those of others. In this invention, this system will be referred to as the “barcode DNA system.” The barcode DNA system is a system defined as a personalized system through arrangements made in certain nucleotide regions, and it enables the retrieval of necessary information from this barcode DNA system when the verification of the information is requested later.

[0024] The barcode DNA system subject to the present invention does not interact with the DNA in the samples obtained from individuals in a way that would cause an adverse effect. It does not possess structures that could interfere with or prevent accurate results during the analysis and testing of the samples. Furthermore, the barcode DNA system combined with the samples is not affected by procedures such as archiving, storage, DNA isolation, the use of saline solutions, or heat applications applied to the samples. In this invention, the preferred application for the individual's biological sample is a blood sample. However, no information is provided in this invention regarding how the sample is collected from the individual. In this respect, the invention does not offer a treatment method or a diagnostic method for individuals. Essentially, the invention presents a biomedical product that enables the labeling of individualspecific samples with a DNA-based barcode system, and a method for obtaining this product.

[0025] As is known, DNA is a data processing mechanism unique to the individual, formed by the combination of the nucleotides adenine (to be referred to as A), guanine (to be referred to as G), cytosine (to be referred to as C), and thymine (to be referred to as T). In this invention, individuals' samples can be labeled with a barcode DNA system that does not exist in the human genome and is created by the substitution of at least one nucleotide, with one part being standard and the other part being specific to the individual.

[0026] The barcode DNA system subject to the invention essentially consists of three parts. The barcode DNA system comprises, in order, a head part, a code part, and an end part.

[0027] In the barcode DNA system, the head part and the end part are the same for each label. The fact that the barcode DNA system provides individual-specific labeling is understood from the differences in the DNA nucleotides present in the code part.

[0028] Accordingly, the DNA sequence subject to the barcode DNA system has a length of 407 bases. Of the 407-base length of the barcode DNA system, 6 bases constitute the code part. The remaining 401 -base length is identical in the barcode systems subject to this invention. The 6-base-long code part enables the generation of 46different combinations of the barcode system. In other words, the barcode DNA system can be applied to as many individuals as the number of combinations.

[0029] The sequence list of the head part of the barcode DNA system subject to the invention is provided as Table 1 .

[0030] Table 1. The head part of the barcode DNA system subject to the invention

[0031] The sequence list of the end part of the barcode DNA system subject to the invention is provided as Table 2.

[0032] Table 2. The end part of the barcode DNA system subject to the invention

[0033] As previously mentioned, the barcode system consists, in order, of the head part whose sequence list is provided in Table 1 , the code part, and the end part whose sequence list is provided in Table 2.

[0034] The individual-specific variable code part, which constitutes the innovative aspect of the invention, has a length of 6 bases within the barcode DNA system. Since an individual-specific sequence arrangement is in question, the code part will be represented by the symbol “N.” The symbol “N” is derived from the English word “any” and is a representation indicating that any of the four nucleotides may be present in this region. For illustrative purposes, different sequences such as ATCCGA and ATAAGG can be used to barcode individuals’ samples.

[0035] In the barcode DNA system, the code part is located at the 201 st, 202nd, 203rd, 204th, 205th, and 206th bases. Each of these nucleotides may be any one of the four variable nucleotides, namely A, T, C, or G. For example, while preparing a barcode DNA system specific to one individual, the sequence may be “NCNNNN,” whereas for another individual, the specific sequence list may be “NGNNNN.” Each of the “N”s indicated here may represent different nucleotides.

[0036] In a different arrangement, even if the same nucleotide is present, it may be located in a different base position. For example, if a barcode DNA system containing the code part ANNNNN is used for labeling one individual, a barcode DNA system containing the code part NANNNN may be used for labeling another individual.

[0037] In this way, in the barcode DNA system subject to the invention, a total of 46barcode systems can be established through the combination of four different nucleotides in a 6-base-long code part. That is, this number of combinations enables labeling for this number of individuals. It is evident that a combination count of 46represents a high labeling capacity for a sample analysis center. The nucleotide sequences shared here, along with the surrounding regions, are important because they include the primer sequences used in the operation of the barcode system.

[0038] The invention also provides a method for obtaining a personalized sequence list of the barcode DNA system. According to this method, the nucleotide sequences forming the barcode DNA system are obtained by molecular cloning.

[0039] In the method subject to the invention, the first process step involves generating viral genome sequence lists that do not show similarity to the human genome and that will be used as a pool for obtaining the barcode DNA system. As the next process step, mutagenesis PCR studies are performed on the obtained DNA sequence, and DNA sequence combinations containing code parts with randomly generated 6-base-long nucleotides are obtained.

[0040] E. coli cells are used for the amplification and preparation of the DNA sequence combinations for use. In the relevant technical field, PCR methods are more commonly used for such amplification processes. In the invention, by using the molecular cloning method as a process step, a procedure that is easier, more controllable, and less costly compared to the PCR method can be achieved. In terms of preserving DNA integrity, the molecular cloning process is more successful compared to laboratory-based methods. Through molecular cloning, a specific DNA sequence can be obtained in bacteria from the DNA sequence combination obtained in the previous process step.

[0041] In this invention, the molecular cloning process step can preferably be carried out using bacteria. In a preferred application, one of the 5-alpha or 10-beta bacterial strains is used for the molecular cloning process. By generating a target sequence list with a defined number of nucleotides from the DNA barcode systems obtained from the mentioned bacterial strains, it becomes possible to obtain a personalized barcode DNA. The obtained specific barcode is amplified in a quantity corresponding to the number of DNA molecules in the sample obtained from the individual, and by adding this barcode DNA into the sample obtained from the individual, a labeling system can be established.

[0042] In this invention, the characterized barcode DNA system includes the head part and the end part, respectively located before and after the 6-base-long code part. For the acquisition of DNAs containing these sequence lists, sequencing approaches such as Sanger, NGS, and / or MG I platforms can be used. In the invention, the barcode DNA system also includes suitable primer regions to allow the application of these sequencing approaches. The scope of protection of the invention is not limited to the method by which the viral genome sequence list, which serves as the raw material of the barcode DNA system, is obtained.

[0043] In this invention, purification is carried out from the DNA pool obtained from viral genome sequence lists in such a way that each will serve as a personalized barcode DNA. The DNAs in the obtained pool are 512 bases in length. These 512- base-long DNAs are subsequently used for the next process steps. As a result of all the procedures performed, the final base length of the obtained barcode DNA systems is 407 bases. One of the main innovative aspects of the invention is also the method that enables these labeling processes. The method subject to the invention comprises the following essential process steps:

[0044] - Formation of DNA fragments and insertion into bacterial plasmids

[0045] The DNAs present as a pool will constitute a mixture carrying potential different barcode DNA system sequences.

[0046] Each DNA in the pool is inserted into a plasmid vector. The plasmid vector may vary depending on the type of bacterium to be used. Subsequently, process steps known in the art that facilitate the integration of DNA fragments into plasmids are applied. Although not detailed in this invention, these process steps may include digestion processes using restriction enzymes or other ligation procedures, as well as transformation steps.

[0047] As a result of these processes, the DNAs in the pool are inserted into the plasmids.

[0048] Each of the aforementioned specific barcode DNA systems is considered as being inserted into a separate plasmid.

[0049] - Colony formation for the bacteria into which the DNAs have been inserted

[0050] In this process step, colony formation is carried out for the bacteria containing DNA. The purpose of this step is to ensure that the bacteria multiply in a culture medium and form a visible colony. The cultures used for colony formation of the bacteria are described below.

[0051] Agar culture can be used as the first culture. The said agar culture contains LB agar with 50 mg / ml ampicillin. The incubation for colony formation is carried out at an ambient temperature in the range of 36-37 °C. The incubation process is preferably performed overnight for at least 12 hours.

[0052] Liquid culture can be used as the second culture. The said liquid culture contains LB with 50 mg / ml ampicillin. The incubation for colony formation is carried out at an ambient temperature in the range of 36-37 °C. The incubation process is preferably performed overnight for at least 12 hours.

[0053] - Selection of bacterial colonies containing DNA fragments

[0054] In the preferred culture media, colonies formed by the bacteria are selected. For colony selection, the barcode region is amplified by the PCR method and sequenced. Samples carrying the correct sequence are selected by analyzing the obtained sequence data. Samples with negative PCR results or those containing deletions, duplications, or repeats are not subjected to further study.

[0055] Accordingly, in this process step, plasmid DNA is isolated from the falcon tubes in which the growth of the colony-forming bacteria is observed, using a silica membrane-based isolation kit. As previously mentioned, the obtained plasmid contains a unique and specific barcode DNA system sequence. Amplification is performed by PCR using primer sequences flanking the barcode sequence on the obtained plasmid DNA, and preferably, the PCR products are sequenced by Next Generation Sequencing (NGS) to determine and record the sequence of the specific barcode DNA system of the obtained colony.

[0056] - Indexing of colonies by PCR method

[0057] Following the selection of bacterial colonies containing DNA fragments, the indexing of the plasmids containing DNA is performed by the PCR method using the following parameters.

[0058] The PCR method is used for the amplification and identification of the selected colonies containing DNA fragments. Accordingly, the targeted DNA is obtained from the bacterial colonies and used as a template for the PCR method. Subsequently, PCR reactions are carried out using the DNA sequence as a template along with designed primers. In this way, the DNA sequences are amplified. Accordingly, the PCR mixture to be used in the PCR method includes the following components:

[0059] - Plasmid DNA in an amount ranging from 5 to 50 ng,

[0060] - Forward and reverse primers in an amount ranging from 1 to 10 pM,

[0061] - dNTP in an amount ranging from 1 to 15 nM,

[0062] - Water in an amount ranging from 20 to 30 pl.

[0063] The PCR parameters applied for the characterized PCR mixture are as follows:

[0064] - a first step at 95 °C for 10 minutes,

[0065] - a second step consisting of the following three stages repeated in 50 cycles:

[0066] • 95 °C for 45 seconds

[0067] • 60 °C for 45 seconds

[0068] • 72 °C for 45 seconds

[0069] - a third step at 72 °C for 10 minutes.

[0070] The PCR products obtained by applying the PCR process steps are visualized using 2% agarose gel electrophoresis. The plasmid DNAs determined to give positive results are purified using magnetic beads. The purified DNA products are quantified using a fluorometric quantification kit.

[0071] Purification of plasmids

[0072] This process step involves the purification or isolation of plasmid DNAs. Various techniques and kits can be used for the purification of DNA. The purified plasmid DNA is dissolved in an appropriate buffer solution. As a result of all these procedures, the barcode DNA system intended for individual-specific use can be obtained. For storage, the barcode DNA systems are dissolved in TE buffer solution after being purified with magnetic beads. As known in the art, the TE buffer solution contains 10 mM Tris and 1 mM EDTA components.

[0073] - Mixing of the obtained barcode DNAs with the samples collected from individuals After the barcode molecules amplified by PCR from validated colony samples are checked by gel electrophoresis, their concentration is determined fluorometrically. The samples are first diluted to 1 ng / pl (with TE dilution buffer solution). Then, they are further diluted to obtain 1000 copies of the barcode DNA system per ml using the formula below. From the resulting 1000 copies / ml barcode solution, 0.5 ml of the barcode DNA system-buffer solution is added to each 1 ml of biological sample.

[0074] DNA molecule copy number = (DNA concentration (ng) x 6.022 x 1023) / (base length of the DNA product x 660 x 109)

[0075] The barcode DNA molecule obtained by means of the method subject to the invention comprises a 6-digit specific genetic code randomly generated with 4 nucleotide variables (Adenine-A, Cytosine-C, Guanine-G, and Thymine-T) thereon, each of the 6-digit random combinations of which can be used as a unique and specific code after cloning processes.

[0076] The sequence list of the barcode DNA system obtained representatively is presented as Table 3.

[0077] Table 3. Sequence list for the sample barcode DNA system In the method subject to the invention, the structures obtained after the cloning step in question can be repeatedly used in the production of barcode DNA molecules by using a PCR device. The scope of protection of the invention is defined in the claims provided in the annex and shall by no means be limited to the examples described in this detailed description. It is evident that a person skilled in the art may develop similar structures in light of the above explanations without departing from the main concept of the invention.

Claims

CLAIMS1 . A barcode DNA system comprising a code part sequence list with a length of 6 bases, characterized by the fact that said code part sequence list has a nucleotide sequence specific to the individual, allowing specific distinction from other samples in analysis centers or during transportation and storage processes when mixed with biological samples belonging to individuals.

2. The barcode DNA system according to claim 1 , characterized in that it comprises a code region sequence list having a nucleotide sequence specific to an individual, and has a head region sequence list up to the said code region sequence list and a tail region sequence list after the code region sequence list.

3. The barcode DNA system according to claim 2, wherein the head region sequence list is SEQ I D : 1 .

4. The barcode DNA system according to claim 2 or claim 3, wherein the tail region sequence list is SEQ ID:2.

5. The barcode DNA system according to any one of the preceding claims, wherein the base length is 407 and the code region consists of the 201 st, 202nd, 203rd, 204th, 205th, and 206th bases.

6. A method for obtaining a barcode DNA system comprising a head region having the sequence list SEQ ID:1 , a code region having a nucleotide sequence specific to an individual located between the head region and a tail region having the sequence list SEQ ID:2, comprising the following process steps:- obtaining a pool of DNAs comprising a randomly arranged code region located between a head region having the sequence list SEQ ID:1 and a tail region having the sequence list SEQ ID:2,insertion of each of the DNAs in the pool into plasmid vectors,- addition of at least one culture to allow colony formation of bacteria containing plasmid vectors with DNA fragments,- selection of bacteria containing DNA fragments that have been colonized by addition to the culture,- amplification of the selected bacteria by PCR method and obtaining the plasmid vector comprising a specific barcode DNA system that can be labeled specifically for individuals,- obtaining the target barcode DNA system by purification of the obtained plasmid vector.

7. The method according to claim 6, characterized in that plasmid vectors belonging to E. coli bacteria are used as the said plasmid vector.

8. The method according to claim 6 or claim 7, characterized in that an agar culture or a liquid culture is used as the culture for colony formation of the bacteria containing plasmid vectors with DNA.

9. The method according to claim 8, characterized in that the said culture is incubated at a temperature of 36-37 °C for at least 12 hours.

10. The method according to any one of claims 6 to 9, characterized in that the following PCR mixture is used for the said PCR process step:- a plasmid vector containing DNA in an amount ranging from 5 to 50 ng,- forward and reverse primers in an amount ranging from 1 to 10 pM, dNTP in an amount ranging from 1 to 15 nM,water in an amount ranging from 20 to 30 pl.11 .The method according to any one of claims 6 to 10, characterized in that the process steps to be applied for the PCR process step are as follows:- a first step at 95 °C for 10 minutes,- a second step consisting of the following three stages repeated in 50 cycles:• 95 °C for 45 seconds• 60 °C for 45 seconds• 72 °C for 45 seconds- a third step at 72 °C for 10 minutes of incubation.

12. The method according to claim 1 1 , characterized in that a gel electrophoresis process step is applied to the plasmid vectors containing DNA that have undergone the PCR process step.

13. The process according to any one of the preceding claims for mixing a barcode DNA system having a nucleotide sequence specifically selected for an individual with a sample belonging to the individual, characterized in that it comprises the following process steps:- dilution of the obtained barcode DNA system with a buffer solution,- addition of the buffer solution-barcode DNA system mixture to the biological sample belonging to the individual.

14. The process according to claim 13, characterized in that TE buffer solution is used as the buffer solution.

15. The process according to claim 13 or claim 14, characterized in that the amount of the barcode DNA system in the buffer solution is 1 ng / pl after the dilution process.

16. The process according to claim 15, characterized in that the dilution process is carried out such that there are 1000 barcode DNA systems in 1 ml of the mixture.

17. The process according to any one of claims 13 to 16, characterized in that 0.5 ml of the barcode DNA system-buffer solution is included for 1 ml of biological sample.

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