Methods for comparing genetic identity, and methods for determining the identity of individuals from which multiple samples originate.

Abstract

To provide a method for comparing genetic identity, and to provide a method for determining the identity of individuals from which multiple samples are derived.SOLUTION: Some embodiments of the present disclosure provide a method for comparing the identities of genes, which includes comparing the identities between human leukocyte antigen genes, short tandem repeat sequences, and killer cell immunoglobulin-like receptor genes of a test sample and a target gene group, and determining that the target gene group is identical to the test gene group of the test sample if comparison results of the human leukocyte antigen genes, comparison results of the short tandem repeat sequences, and comparison results of the killer cell immunoglobulin-like receptor genes are all identical. Some embodiments of the present disclosure further provide a method for determining the identities of individuals from whom multiple samples are derived.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to a method for comparing gene identities and a method for determining the identities of individuals from which a plurality of samples are derived.

Background Art

[0002] In cell therapy or organ transplantation, the higher the gene identity between the acting cells and the original individual cells, the lower the risk of causing immune rejection. When the acting cells and the original individual cells are derived from the same individual, the risk of immune rejection can be minimized. Therefore, when an individual is in a healthy state, individual cells can be cryopreserved in advance for use when cell therapy or organ transplantation needs to be performed later. However, since a large number of cells derived from different individuals are usually stored in a cell cryopreservation bank at the same time, misidentifying the acting cells used for treatment will cause a serious immune rejection reaction in the individual.

[0003] Therefore, in order to reduce the risk of immune rejection, it is necessary to confirm whether the acting cells (hereinafter referred to as test samples) and the original individual cells (hereinafter referred to as target samples) are derived from the same individual before treatment and then continue the subsequent treatment.

[0004] However, conventional gene comparison methods have limited accuracy (for example, when identifying short tandem repeats (STR) and the genes are more than 80% identical, they are determined to be from the same individual), and there is still a significant risk of misjudgment.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Therefore, how to improve the accuracy of the method for comparing gene identities and determining the identities of individuals from which a plurality of samples are derived is a problem to be solved.

Means for Solving the Problems

[0006] Some embodiments of this disclosure include the steps of: providing a group of target genes; providing a test sample comprising a first test sample, a second test sample, and a third test sample; comparing the identity between the human leukocyte antigen (HLA) gene of the first test sample and the human leukocyte antigen gene of the group of target genes and obtaining the comparison result of the human leukocyte antigen gene; comparing the identity between the short tandem repeat sequence (STR) of the second test sample and the short tandem repeat sequence of the group of target genes and obtaining the comparison result of the short tandem repeat sequence; and providing the killer-cell immunoglobulin-like receptor (KIR) of the third test sample. The present invention provides a method for comparing the identity of genes, comprising the steps of: comparing the identity between a receptor (KIR) gene and the killer cell immunoglobulin-like receptor gene of a target gene group and obtaining the comparison result of the killer cell immunoglobulin-like receptor gene; and determining that the target gene group is identical to the test gene group of the test sample if the comparison result of the human leukocyte antigen gene, the comparison result of the short tandem repeat sequence, and the comparison result of the killer cell immunoglobulin-like receptor gene are all identical.

[0007] In some embodiments, the first test sample includes blood.

[0008] In some embodiments, the step of comparing the identity between the human leukocyte antigen gene of a first test sample and the human leukocyte antigen gene of a group of target genes includes the steps of extracting and amplifying the human leukocyte antigen gene of the first test sample and analyzing and comparing the identity between the human leukocyte antigen gene of the first test sample and the human leukocyte antigen gene in the group of target genes using next-generation sequencing.

[0009] In some embodiments, the step of comparing the identity between the human leukocyte antigen gene of a first test sample and the human leukocyte antigen gene of a group of target genes includes the step of comparing the identity of HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, or combinations thereof.

[0010] In some embodiments, the step of comparing the identity between the human leukocyte antigen gene of a first test sample and the human leukocyte antigen gene of a group of target genes includes the steps of comparing the identity between the coding region in the human leukocyte antigen gene of the first test sample and the coding region in the human leukocyte antigen gene of a group of target genes, and the steps of comparing the identity between the non-coding region in the human leukocyte antigen gene of the first test sample and the non-coding region in the human leukocyte antigen gene of a group of target genes.

[0011] In some embodiments, the second test sample includes human tissue cells.

[0012] In some embodiments, the step of comparing the identity between a short tandem repeat sequence of a second test sample and a short tandem repeat sequence of a group of target genes includes the steps of extracting a short tandem repeat sequence from the second test sample and analyzing and comparing its identity with that of a short tandem repeat sequence in a group of target genes using a sequencing typing method.

[0013] In some embodiments, the step of comparing the identity between the short tandem repeat sequence of a second test sample and the short tandem repeat sequences of the target gene group includes comparing the identity of genes at the loci D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, the amelogenin gene, D5S818, FGA, or combinations thereof.

[0014] In some embodiments, the third test sample includes blood.

[0015] In some embodiments, the step of comparing the identity between the killer cell immunoglobulin-like receptor gene of a third test sample and the killer cell immunoglobulin-like receptor gene of a group of target genes includes the steps of: extracting the killer cell immunoglobulin-like receptor gene of the third test sample; and analyzing and comparing the identity between the killer cell immunoglobulin-like receptor gene of the third test sample and the killer cell immunoglobulin-like receptor gene of a group of target genes using polymerase chain reaction-sequence-specific oligonucleotide probes (PCR-SSOP), polymerase chain reaction-sequence-specific primers (PCR-SSP), sequence-based typing (SBT), or a combination thereof.

[0016] In some embodiments, the step of comparing the identity between the killer cell immunoglobulin-like receptor gene of a third test sample and the killer cell immunoglobulin-like receptor gene of a group of target genes includes the step of comparing the identity of genes 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, or combinations thereof.

[0017] Some embodiments of this disclosure further provide a method for determining the identity of an individual from which multiple samples originate, comprising the steps of: providing a test sample and a target sample; comparing the identity of human leukocyte antigen genes between the test sample and the target sample and obtaining the comparison result of the human leukocyte antigen genes; comparing the identity of short tandem repeat sequences between the test sample and the target sample and obtaining the comparison result of the short tandem repeat sequences; comparing the identity of killer cell immunoglobulin-like receptor genes between the test sample and the target sample and obtaining the comparison result of the killer cell immunoglobulin-like receptor genes; and determining that the individual from which the test sample and the target sample originate is the same if the comparison result of the human leukocyte antigen genes, the comparison result of the short tandem repeat sequences, and the comparison result of the killer cell immunoglobulin-like receptor genes are all the same.

[0018] In some embodiments, the test sample and the target sample include blood.

[0019] In some embodiments, the step of comparing the identity of human leukocyte antigen genes between a test sample and a target sample includes the steps of extracting and amplifying the human leukocyte antigen genes of the test sample and the target sample, and analyzing and comparing the identity of the human leukocyte antigen genes of the test sample and the target sample using next-generation sequencing.

[0020] In some embodiments, the step of comparing the identity of human leukocyte antigen genes between a test sample and a target sample includes the step of comparing the identity of HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, or combinations thereof.

[0021] In some embodiments, the step of comparing the identity of human leukocyte antigen genes between a test sample and a target sample includes the steps of comparing the identity of the coding region in the human leukocyte antigen gene of the test sample with the coding region in the human leukocyte antigen gene of the target sample, and comparing the identity of the non-coding region in the human leukocyte antigen gene of the test sample with the non-coding region in the human leukocyte antigen gene of the target sample.

[0022] In some embodiments, the step of comparing the identity of short tandem repeat sequences between a test sample and a target sample includes the steps of extracting the short tandem repeat sequences of the test sample and the target sample, and analyzing and comparing the identity of the short tandem repeat sequences of the test sample and the target sample using a sequencing typing method.

[0023] In some embodiments, the step of comparing the identity of short tandem repeat sequences between a test sample and a target sample includes comparing the identity of genes at the loci D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, the amelogenin gene, D5S818, FGA, or combinations thereof.

[0024] In some embodiments, the step of comparing the identity of killer cell immunoglobulin-like receptor genes between a test sample and a target sample includes the steps of: extracting the killer cell immunoglobulin-like receptor genes from the test sample and the killer cell immunoglobulin-like receptor genes from the target sample; and analyzing and comparing the identity of the killer cell immunoglobulin-like receptor genes from the test sample and the killer cell immunoglobulin-like receptor genes from the target sample using a sequence-specific oligonucleotide probe method, a polymerase chain reaction-sequence-specific primer method, a sequencing typing method, or a combination thereof.

[0025] In some embodiments, the step of comparing the identity of killer cell immunoglobulin-like receptor genes between a test sample and a target sample includes comparing the identity of genes of 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, or combinations thereof.

Brief Description of Drawings

[0026] To make the above and other objects, features, advantages and embodiments of the present invention more understandable, the description of the accompanying drawings is as follows. [Figure 1] It is a flowchart of a method for comparing the identity of genes in some embodiments of the present disclosure. [Figure 2] It is a flowchart of a method for determining the identity of individuals from which multiple samples in some embodiments of the present disclosure are derived.

Modes for Carrying Out the Invention

[0027] To describe the present invention in more detail and completely, the embodiments and specific examples of the present invention will be described in detail below, but these are not the only forms for implementing or applying the specific examples of the present invention. Each example disclosed below may be combined or substituted with each other without further description or explanation if beneficial, and other examples may be added to one example. In the following description, many specific details are described in detail to enable the reader to fully understand the following examples. However, the embodiments of the present invention can be implemented without these specific details.

[0028] In this specification, unless otherwise specified in the text, the articles "one" and "the" generally refer to one or more. To further understand, the terms "include," "contain," "have" and similar terms used herein specify the features, areas, integers, processes, operations, elements and / or components described, but do not exclude other features, areas, integers, processes, operations, elements, components and / or groups thereof.

[0029] The methods disclosed herein will be described below using a series of operations or steps, but the procedures shown in these operations or steps should not be construed as limiting the invention. For example, some operations or steps may be performed in different procedures and / or simultaneously with other steps. Furthermore, it is not necessary to perform all operations, steps and / or features in order to realize embodiments of the invention. Moreover, each operation or step described herein may include multiple sub-operations or steps.

[0030] In this specification, polymerase chain reaction-sequence-specific oligonucleotide method (PCR-SSOP) refers to a method in which a probe labeled with an isotope or non-radioactive material is hybridized with fragments of a group of test genes amplified by a polymerase chain reaction (PCR), and the genotype of the test gene group is determined via the signal that matches the hybridization.

[0031] In this specification, polymerase chain reaction-sequence-specific primer method (PCR-SSP) refers to a method that uses PCR to amplify genes with specific primers, and then determines the genotype of the tested gene group based on whether or not amplification has occurred.

[0032] In this specification, sequencing-based typing (SBT) refers to a method of directly determining the sequence of a gene, such as Sanger SBT (SSBT) or next-generation sequencing (NGS).

[0033] Some embodiments of this disclosure compare the identity of human leukocyte antigen (HLA) genes, short tandem repeat sequences (STR), and killer-cell immunoglobulin-like receptor (KIR) genes in test samples and target samples to confirm whether the target gene group is identical to the test gene group, and to determine whether the test sample and target sample originate from the same individual. By comparing highly polymorphic triple genes, it is as if the test sample and target sample are encrypted with a gene password lock, minimizing the probability of mistaking the test sample (the probability of identical results is 1 in 10 billion).

[0034] First, please refer to Figure 1. A method 100 for comparing gene identity is provided, which includes steps S110, S120, S130, S140, S150, and S160.

[0035] Step S110 provides a group of target genes.

[0036] In some embodiments, the target gene group consists of the gene sequences of the target sample and includes human HLA genes, STR, and KIR genes. In some embodiments, the HLA genes of the target gene group include the genes for HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1. In some embodiments, the STR of the target gene group includes the sequences for loci D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, the amelogenin gene, D5S818, and FGA. In some embodiments, the KIR genes of the target gene group include the genes 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, and 3DP1. It should be emphasized that the gene fragments in HLA, STR, and KIR are fragments with high polymorphism in HLA, STR, and KIR, respectively, which reduces the probability (approximately 1 in 10 billion) of a gene in another sample being completely identical to the target gene group when compared later, thereby improving the accuracy of the comparison. In some embodiments, the target sample includes blood, human tissue cells, or a combination thereof.

[0037] Step S120 provides a test sample comprising a first test sample, a second test sample, and a third test sample.

[0038] In some embodiments, the first test sample comprises blood (primarily used to provide lymphocytes) and is used to analyze HLA genes. In some embodiments, the second test sample comprises human tissue cells and is used to analyze STR. For example, human tissue cells include oral cells, blood, hair, placenta, umbilical cord, amniotic fluid, saliva, or a combination thereof. In some embodiments, the third test sample comprises blood (primarily used to provide natural killer cells) and is used to analyze KIR genes. The type of test sample substantially corresponds to the type of target sample.

[0039] Step S130 compares the identity between the HLA gene of the first test sample and the HLA gene of the target gene group, and obtains the comparison results for the HLA genes.

[0040] In some embodiments, step S130 includes the steps of extracting and amplifying the HLA gene from the first test sample, and analyzing and comparing the identity of the HLA gene from the first test sample with the HLA gene in the target gene group using NGS.

[0041] In some embodiments, high resolution of up to 8 orders of magnitude can be achieved by using NGS. Specifically, the first and second digits (first region) represent HLA serological typing or allelic genomes, the third and fourth digits (second region) represent alleles with amino acid mutations in the coding region, the fifth and sixth digits (third region) represent alleles without amino acid mutations in the coding region (synonymous DNA substitution), and the seventh and eighth digits (fourth region) represent alleles with base substitutions in the non-coding region.

[0042] Specifically, step S130 includes a step of comparing the identity of the coding region in the HLA gene of the first test sample with the coding region in the HLA gene of the target gene group, and a step of comparing the identity of the non-coding region in the HLA gene of the first test sample with the non-coding region in the HLA gene of the target gene group. It should be emphasized that by further analyzing and comparing the gene sequences of the non-coding regions compared to analyzing only the coding region, it is possible to improve the accuracy of determining gene identity, and by incorporating the comparison results of the non-coding regions into the reference, it is possible to improve the success rate of treatment when performing cell therapy or organ transplantation.

[0043] In some embodiments, step S130 includes comparing the identity of the HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, or combinations thereof, where these subtypes are highly polymorphic. As can be understood, HLA genes are the category of genes with the highest polymorphism among human genes, and the accuracy of gene comparison can be further improved by simultaneously selecting and comparing the highly polymorphic subtypes among the HLA genes.

[0044] In some embodiments, the HLA gene comparison results indicate whether the HLA gene of the first test sample is identical to each subtype of the target gene group's HLA. The HLA gene comparison results are considered identical if all measured HLA subtype results are identical.

[0045] Step S140 compares the identity between the short tandem repeat sequence (STR) of the second test sample and the short tandem repeat sequence (STR) of the target gene group, and obtains the STR comparison results.

[0046] In some embodiments, step S140 includes the steps of extracting STRs from a second test sample and analyzing and comparing the identity of the STRs from the second test sample with the STRs in the target gene group using SBT. Compared to PCR-SSP or PCR-SSOP, SBT can achieve higher comparison accuracy because it directly sequences the genes.

[0047] In some embodiments, step S140 includes comparing the identity of the loci D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, the amelogenin gene, D5S818, FGA, or combinations thereof, where these loci are highly polymorphic. As can be understood, STR is a category of highly polymorphic genes in human genes, and the accuracy of gene comparison can be improved by further selecting and comparing highly polymorphic loci within the STR genes.

[0048] Step S150 compares the identity between the KIR gene of the third test sample and the KIR gene of the target gene group, and obtains the comparison results for the KIR genes.

[0049] In some embodiments, step S150 includes the steps of extracting the KIR gene from a third test sample and analyzing and comparing the identity of the KIR gene from the third test sample with the KIR gene in the target gene group using PCR-SSOP, PCR-SSP, SBT, or a combination thereof.

[0050] In some embodiments, multiple analytical methods can be combined and compared to improve detection accuracy. For example, by first performing PCR-SSOP and PCR-SSP, which take less time, and comparing whether the results of the two test methods are identical, errors present in individual methods can be reduced. In another example, when designing primers for gene fragments is difficult, PCR-SSOP can be selected and combined with SBT, which has high sequence resolution, to compare whether the results of the two test methods are identical. This can save primer design time and overcome the limitations on gene detection segments that exist when using PCR-SSP due to the difficulty of primer design.

[0051] In some embodiments, step S150 includes comparing the identity of genes 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, or combinations thereof, where these gene fragments are highly polymorphic genes. As can be understood, KIR genes are a category of highly polymorphic genes in human genes and are highly associated with immune responses. Further simultaneous selection and comparison of highly polymorphic gene segments within the KIR genes can improve the accuracy of gene comparison and reduce the risk of immune rejection during treatment.

[0052] Please refer to Figure 2. This disclosure further provides a method 200 for determining the identity of an individual from which multiple samples originate, including steps S210 to S250. Step S210 provides a test sample and a target sample. In some embodiments, the analysis of HLA genes, STR, and KIR genes can be completed when all test and target samples are blood.

[0053] Step S220 compares the identity of HLA genes between the test sample and the target sample and obtains the HLA gene comparison results. Step S230 compares the identity of STR between the test sample and the target sample and obtains the STR comparison results. Step S240 compares the identity of KIR genes between the test sample and the target sample and obtains the KIR gene comparison results. Step S250 determines that the test sample and the target sample originate from the same individual if the HLA comparison results, STR comparison results, and KIR gene comparison results are all identical. Step S220 substantially corresponds to Step S130, Step S230 substantially corresponds to Step S140, Step S240 substantially corresponds to Step S150, Step S250 substantially corresponds to Step S160, and the analytical and comparison methods can be found by referring to the above and will not be repeated here.

[0054] Based on the above, Method 200 can improve the accuracy of interpretation by analyzing and comparing three gene types (HLA gene, STR gene, KIR gene) that exhibit high polymorphism, thereby reducing the probability of identical gene comparison results to 1 in 10 billion. Therefore, when the gene comparison results are identical, it can be determined that the individual from which the test sample and the target sample originated is the same, and this can be used for subsequent treatment.

[0055] To further illustrate the methods for comparing genetic identity and determining the identity of individuals from multiple samples provided by various embodiments of this disclosure, the following actions are taken. Note that the following examples are provided for illustrative purposes only and are not intended to limit the invention.

[0056] The following examples illustrate three different procedures for determining genetic identity. Since the genes of the target sample and the test sample can be analyzed using the same method, for simplicity, the following examples will all be representative of the genetic analysis process of the test sample. It will be shown that the HLA, STR, and KIR genes of the target sample can be analyzed in advance using the same method as the test sample before the cells are cryopreserved.

[0057] 1. HLA gene identity

[0058] The HLA gene is the most polymorphic system in humans, and the probability of two individuals having completely identical HLA genes is extremely low (depending on race, the probability of two individuals having completely identical HLA genes ranges from 1 in 2 billion to 1 in 200,000). Therefore, by selecting HLA genes and comparing their genetic identity, the accuracy of determining genetic identity between a target sample and a test sample can be improved. The following is an example of the analysis process.

[0059] First, blood was provided as the test sample.

[0060] Next, using TBG Diagnostics' HLAssure SE human leukocyte antigen SBT genotyping reagents (HLAssure SE A Locus SBT Kit, product number 50110; HLAssure SE B Locus SBT Kit, product number 50210; HLAssure SE C Locus SBT Kit, product number 50410; HLAssure SE DRB1-EX Locus SBT Kit, product number 50350; HLAssure SE DQB1 Locus SBT Kit, product number 50510), human leukocyte antigen genes were extracted and amplified from the test sample. After creating a library of human leukocyte antigen genes, an HLA high-resolution typing sequencer (brand: Applied Biosystems) was used to perform the analysis. TM Using the 3730xl DNA Analyzer (product number: A41046), the sequences of HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 can be analyzed by NGS, achieving a sequencing resolution of 8 digits and including the genotype of both coding and non-coding regions.

[0061] Next, we compared whether the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 genes in the target sample and the test sample were completely identical, and obtained the results of the HLA gene comparison.

[0062] It should be explained that the resolution of NGS reflects whether or not the gene sequence can be read completely. Taking HLA-A as an example, based on sequence differences, A01 can be subdivided into A01:01(GGATCATC), A01:02(GGATGAAG), A01:03(GGATCTAG), etc. Using low-resolution analysis, for example, analyzing to two orders of magnitude, only the first four gene sequences such as A01(GGATXXXX), A02(CTGGXXXX), A03(ATCGXXXX) can be compared, while the latter four are all ignored, thereby rapidly detecting whether or not a result is positive. However, using low-resolution analysis increases the probability of rejection or death in cell therapy or organ transplantation, even if HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ all match.

[0063] Therefore, compared to sequencing with a resolution of 2 or 4 digits (as is generally known, if sequences are detected up to 4 digits and compared with more than 80% identical, they are judged to be individuals of the same origin), the sequence resolution of the NGS of the present invention is improved to 8 digits (for example, reading HLA-A A*01:01:01:01), and the genes must be completely identical, thereby improving the accuracy of interpreting HLA sequence identity and reducing subsequent rejection reactions to treatment.

[0064] 2. Identity of STR

[0065] The gene region of STR consists of base pairs 3 to 7 in length. STRs are widely distributed throughout the human genome and exhibit high diversity. Therefore, by selecting STRs and performing identity comparisons, the accuracy of determining genetic identity between the target sample and the test sample can be improved. The following is an example of the analysis process.

[0066] First, human tissue cells (e.g., oral cells or blood) were provided as test samples.

[0067] Next, a DNA extraction kit (Brand: Invitrogen, Name: PureLink) TMSTRs were extracted from the test samples using the Genomic DNA Mini Kit (product number: K182001).

[0068] Amplification kit (Brand: Applied Biosystems, Name: AmpFLSTR) (R) Identifiler (R) The extracted STRs were amplified using the PCR Amplification Kit (product number: 4322288).

[0069] Using a sequence analyzer (brand: Applied Biosystems, name: SeqStudio Genetic Analyzer, product number: A35644), the sequences of the following loci in STR were determined by sequencing-based typing (SBT): D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, the amelogenin gene, D5S818, and FGA.

[0070] Next, we compared whether the genes at the aforementioned locus of STR in the target sample and the test sample were completely identical, and obtained the comparison results for STR.

[0071] It should be explained that, compared to selecting other loci of STR, the combination of STR loci of the present invention can improve the accuracy of gene identity determination to 99% by selecting a region within STR that has a high degree of genetic diversity.

[0072] 3. The Identity of KIR

[0073] KIR genes are a group of genes located on human chromosome 19. The presence of KIR genes allows KIR proteins to be expressed in natural killer cells, and KIR proteins play a crucial role in the human immune response. While there are a total of 17 KIR genes, not everyone expresses all of them. Furthermore, different combinations of KIR genes can combine to form single units of different KIR genes, resulting in a high degree of genetic polymorphism. In organ transplantation, when HLA genes are identical, recipients and donors possess the same single unit of KIR gene, further improving transplant success rates and reducing the probability of acute myeloid leukemia (AML). Therefore, having identical KIR genes (originating from the same individual) contributes to improving the success rate of subsequent cell therapy or organ transplantation.

[0074] The following is an example of the analysis process.

[0075] First, blood was provided as the test sample.

[0076] Next, we have the KIR typing kit (name: Explobe) from TBG Biotechnology Corp. TM Using the KIR Typing Kit (product number: 69010), PCR was performed on a PCR machine (brand: Applied Biosystems, name: 7500 Real-Time PCR system) using the PCR-SSP method. The presence or absence of the desired gene fragments in the test sample was confirmed by performing PCR on 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, and 3DP1 in the KIR using sequence-specific primers.

[0077] To ensure clarity, the methods used for analyzing the KIR genotype described above are all applicable to the examples and include, but are not limited to, PCR-SSP, PCR-SSOP, and SBT. Furthermore, the accuracy of detection can be improved by combining multiple sequencing methods simultaneously as needed.

[0078] Next, we compared whether the distribution of the KIR gene blocks (which is the sequencing result in the SBT method) in the target sample and the test sample were identical, and obtained the comparison results for the KIR genes.

[0079] 4. Determination of the genetic identity of the sample

[0080] By comparing the three gene sequences of Example 1 (HLA), Example 2 (STR), and Example 3 (KIR), which exhibit the high degree of polymorphism described above, we were able to reduce the probability that the target gene group is identical to the test gene group to approximately 1 in 10 billion. Therefore, if the three comparison results for HLA, STR, and KIR of the target sample and the test sample are all identical, theoretically only one person in the entire world population would be a match. This is equivalent to locking three genes between the samples, and because the probability of genetic identity is extremely low, we ensured that when the genes are identical, the individuals from which they originate are the same.

[0081] Therefore, by comparing the above examples, if the results between the test sample and the target sample are identical, it can be determined that the test gene group in the test sample is identical to the target gene group in the target sample. This indicates that the individuals from which the test sample and the target sample originate are the same, and the test sample can be used for subsequent treatment.

[0082] While the embodiments described above limit the scope of this disclosure, those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure. Therefore, the scope of protection of this disclosure shall be determined by the scope of the patent application to be attached later. [Explanation of symbols]

[0083] 100, 200: Method S110, S120, S130, S140, S150, S160, S210, S220, S230, S240, S250: Engineering

Claims

1. A method for comparing the genetic identity between a group of target genes in a target sample and a group of test genes in a test sample, A step of providing the target sample containing the aforementioned target gene group, A step of providing a test sample including a first test sample, a second test sample, and a third test sample, The process involves comparing the identity between the human leukocyte antigen gene of the first test sample and the human leukocyte antigen gene of the target gene group, and obtaining the comparison results of the human leukocyte antigen genes. The process involves comparing the identity between the short tandem repeat sequence of the second test sample and the short tandem repeat sequences of the target gene group, and obtaining the comparison results of the short tandem repeat sequences. The process involves comparing the identity between the killer cell immunoglobulin-like receptor gene of the third test sample and the killer cell immunoglobulin-like receptor gene of the target gene group, and obtaining the comparison results for the killer cell immunoglobulin-like receptor genes. The step of determining that the target gene group is the same as the test gene group of the test sample if the comparison results of the human leukocyte antigen gene, the comparison results of the short tandem repeat sequence, and the comparison results of the killer cell immunoglobulin-like receptor gene are all the same, Methods that include...

2. The method according to claim 1, wherein the first test sample includes blood.

3. The step of comparing the identity between the human leukocyte antigen gene of the first test sample and the human leukocyte antigen gene of the target gene group is: The process involves extracting and amplifying the human leukocyte antigen gene from the first test sample, A step of using a next-generation sequencing method to compare the identity of the human leukocyte antigen gene in the first test sample with the human leukocyte antigen gene in the target gene group, The method according to claim 1, including the method described in claim 1.

4. The method according to claim 1, wherein the step of comparing the identity between the human leukocyte antigen gene of the first test sample and the human leukocyte antigen gene of the target gene group includes the step of comparing the identity of genes for HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, or combinations thereof.

5. The step of comparing the identity between the human leukocyte antigen gene of the first test sample and the human leukocyte antigen gene of the target gene group is: A step of comparing the identity between the coding region in the human leukocyte antigen gene of the first test sample and the coding region in the human leukocyte antigen gene of the target gene group, A step of comparing the identity between the non-coding region in the human leukocyte antigen gene of the first test sample and the non-coding region in the human leukocyte antigen gene of the target gene group, The method according to claim 1, including the method described in claim 1.

6. The method according to claim 1, wherein the second test sample comprises human tissue cells.

7. The step of comparing the identity between the short tandem repeat sequence of the second test sample and the short tandem repeat sequence of the target gene group is: The steps include extracting the short tandem repeat sequence from the second test sample, A step of using a sequencing typing method to compare the identity of the short tandem repeat sequence of the second test sample with the short tandem repeat sequence in the target gene group, The method according to claim 1, including the method described in claim 1.

8. The method according to claim 1, wherein the step of comparing the identity between the short tandem repeat sequence of the second test sample and the short tandem repeat sequence of the target gene group includes the step of comparing the identity of genes at the locus D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, the amelogenin gene, D5S818, FGA, or combinations thereof.

9. The method according to claim 1, wherein the third test sample includes blood.

10. The step of comparing the identity between the killer cell immunoglobulin-like receptor gene of the third test sample and the killer cell immunoglobulin-like receptor gene of the target gene group is as follows: The process involves extracting the killer cell immunoglobulin-like receptor gene from the third test sample, A step of comparing the identity of the killer cell immunoglobulin-like receptor gene of the third test sample with the killer cell immunoglobulin-like receptor gene in the target gene group, using a polymerase chain reaction-sequence-specific oligonucleotide method, a polymerase chain reaction-sequence-specific primer method, a sequencing typing method, or a combination thereof, The method according to claim 1, including the method described in claim 1.

11. The method according to claim 1, wherein the step of comparing the identity between the killer cell immunoglobulin-like receptor gene of the third test sample and the killer cell immunoglobulin-like receptor gene of the target gene group includes the step of comparing the identity of genes 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, or combinations thereof.

12. A method for determining the identity between an individual from which a test sample originates and an individual from which a target sample originates, The steps include providing the aforementioned test sample and the aforementioned target sample, A step of comparing the identity of human leukocyte antigen genes between the test sample and the target sample, and obtaining the comparison results of human leukocyte antigen genes, A step of comparing the identity of the short tandem repeat sequences between the test sample and the target sample, and obtaining the comparison results of the short tandem repeat sequences. A step of comparing the identity of killer cell immunoglobulin-like receptor genes between the test sample and the target sample, and obtaining the comparison results of the killer cell immunoglobulin-like receptor genes, The step of determining that the individual from which the test sample originates and the individual from which the target sample originates are the same if the comparison results of the human leukocyte antigen gene, the comparison results of the short tandem repeat sequence, and the comparison results of the killer cell immunoglobulin-like receptor gene are all the same. Methods that include...

13. The method according to claim 12, wherein the test sample and the target sample include blood.

14. The step of comparing the identity of the human leukocyte antigen gene between the test sample and the target sample is as follows: A step of extracting and amplifying the human leukocyte antigen gene from the test sample and the human leukocyte antigen gene from the target sample, A step of using next-generation sequencing to compare the identity of the human leukocyte antigen gene of the test sample with the human leukocyte antigen gene of the target sample, The method according to claim 12, including the method described in claim 12.

15. The method according to claim 12, wherein the step of comparing the identity of the human leukocyte antigen gene between the test sample and the target sample includes the step of comparing the identity of the genes HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, or combinations thereof.

16. The step of comparing the identity of the human leukocyte antigen gene between the test sample and the target sample is as follows: A step of comparing the identity between the coding region in the human leukocyte antigen gene of the test sample and the coding region in the human leukocyte antigen gene of the target sample, A step of comparing the identity of the non-coding region in the human leukocyte antigen gene of the test sample with the non-coding region in the human leukocyte antigen gene of the target sample, The method according to claim 12, including the method described in claim 12.

17. The step of comparing the identity of the short tandem repeat sequence between the test sample and the target sample is as follows: A step of extracting the short tandem repeat sequence from the test sample and the short tandem repeat sequence from the target sample, A step of comparing the identity of the short tandem repeat sequence of the test sample with the short tandem repeat sequence of the target sample using a sequencing typing method, The method according to claim 12, including the method described in claim 12.

18. The method according to claim 12, wherein the step of comparing the identity of the short tandem repeat sequences between the test sample and the target sample includes the step of comparing the identity of genes at the loci D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, the amelogenin gene, D5S818, FGA, or combinations thereof.

19. The step of comparing the identity of the killer cell immunoglobulin-like receptor gene between the test sample and the target sample is as follows: A step of extracting the killer cell immunoglobulin-like receptor gene from the test sample and the killer cell immunoglobulin-like receptor gene from the target sample, A step of comparing the identity of the killer cell immunoglobulin-like receptor gene of the test sample with the killer cell immunoglobulin-like receptor gene of the target sample using a sequence-specific oligonucleotide probe method, a polymerase chain reaction-sequence-specific primer method, a sequencing-typeding method, or a combination thereof, The method according to claim 12, including the method described in claim 12.

20. The method according to claim 12, wherein the step of comparing the identity of the killer cell immunoglobulin-like receptor gene between the test sample and the target sample includes the step of comparing the identity of the genes 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, or combinations thereof.

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