Method for comparing genetic identity and method for determining identity of individuals from which multiple samples are derived

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 methods for comparing genetic identity and determining the identity of individuals from which multiple samples are derived. [Background technology]

[0002] In cell therapy or organ transplantation, the higher the genetic identity between the working cells and the cells of the original individual, the lower the risk of immune rejection. Therefore, if the working cells and the cells of the original individual are derived from the same individual, the risk of immune rejection can be minimized. Therefore, if an individual is healthy, the individual's cells can be cryopreserved in advance for use in the event that cell therapy or organ transplantation is required later. However, since a cell cryopreservation facility typically stores a large number of cells derived from different individuals at the same time, mistaking the working cells used for treatment can cause a serious immune rejection reaction in the individual.

[0003] Therefore, to reduce the risk of immune rejection, it is necessary to confirm before treatment whether the active cells (hereinafter referred to as the test sample) and the original individual's cells (hereinafter referred to as the target sample) are derived from the same individual before continuing with subsequent treatment.

[0004] However, conventional genetic comparison methods have limited accuracy (e.g., identification of short tandem repeats (STRs), in which genes with 80% or more identity are determined to be the same individual), and there is still a significant risk of misidentification. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, how to improve the accuracy of methods for comparing genetic identity and determining the identity of the individuals from whom multiple samples are derived is a problem that needs to be solved. [Means for solving the problem]

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

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

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

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

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

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

[0012] In some embodiments, comparing the identity between the short tandem repeat sequences of the second test sample and the short tandem repeat sequences of the target gene group comprises extracting the short tandem repeat sequences of the second test sample and analyzing and comparing the identity between the short tandem repeat sequences of the second test sample and the short tandem repeat sequences in the target gene group using a sequence typing method.

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

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

[0015] In some embodiments, 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 comprises 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 in the target gene group 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, comparing the identity between the killer cell immunoglobulin-like receptor genes of the third test sample and the killer cell immunoglobulin-like receptor genes of the target gene group 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.

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

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

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

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

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

[0022] In some embodiments, comparing the identity of short tandem repeat sequences between the test sample and the target sample comprises 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 sequence-based typing method.

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

[0024] In some embodiments, the step of comparing the identity of the killer cell immunoglobulin-like receptor gene between the test sample and the target sample includes the steps of extracting the killer cell immunoglobulin-like receptor gene of the test sample and the killer cell immunoglobulin-like receptor gene of the target sample, and analyzing and comparing the identity of the killer cell immunoglobulin-like receptor gene of the test sample and 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 typing method by sequencing, or a combination thereof.

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

[0026] To make the above and other objects, features, advantages and embodiments of the present invention more comprehensible, reference is made to the accompanying drawings as follows: [Figure 1] 1 is a flowchart of a method for comparing gene identity in some embodiments of the present disclosure. [Figure 2] 1 is a flowchart of a method for determining the identity of individuals from which multiple samples are derived in some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0027] In order to provide a more detailed and complete description of the present invention, the following describes in detail the embodiments and specific examples of the present invention, but these are not the only ways to implement or apply the specific examples of the present invention. The examples disclosed below may be combined with or substituted for each other, if beneficial, without further description or explanation, and one example may be added to another example. In the following description, numerous specific details are described in detail to allow the reader to fully understand the following examples. However, the present invention can be implemented without these specific details.

[0028] As used herein, "a," "an," and "the" can generally refer to one or more, unless the context clearly indicates otherwise. It will be further understood that, as used herein, the terms "comprise," "include," "have," and similar terms specify stated features, regions, integers, steps, operations, elements, and / or components, but do not exclude other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0029] The methods disclosed herein are described below using a series of operations or steps, but the order in which these operations or steps are presented should not be construed as limiting the present invention. For example, some operations or steps may be performed in different sequences and / or simultaneously with other steps. Also, not all operations, steps, and / or features need to be performed to achieve an embodiment of the present invention. Furthermore, each operation or step described herein may include multiple sub-operations or steps.

[0030] As used herein, polymerase chain reaction-sequence-specific oligonucleotide method (PCR-SSOP) refers to hybridization of isotopically or non-radioactively labeled probes with fragments of test gene groups amplified by polymerase chain reaction (PCR), and determination of the genotypes of the test gene groups based on hybridization match signals.

[0031] As used herein, the polymerase chain reaction-sequence-specific primer method (PCR-SSP) refers to the use of PCR to amplify genes with specific primers, and then determining the genotype of the test gene group based on the presence or absence of amplification.

[0032] As used herein, sequence-by-sequence typing (SBT) refers to a method of directly sequencing a gene, such as found in Sanger SBT (SSBT) or Next Generation Sequencing (NGS).

[0033] Some embodiments of the present disclosure compare the identity of human leukocyte antigen (HLA) genes, short tandem repeats (STRs), and killer-cell immunoglobulin-like receptor (KIR) genes between the test and target samples to determine whether the target genes are identical to the test genes and whether the test and target samples are from the same individual. Comparing highly polymorphic triplet genes creates a genetic password lock between the test and target samples, minimizing the chance of mistaking the test sample (the chance of identical results is 1 in 10 billion).

[0034] Please refer first to Figure 1. A method 100 for comparing the identity of genes is provided, including 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 is a gene sequence of a target sample and includes human HLA genes, STRs, and KIR genes. In some embodiments, the HLA genes of the target gene group include HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 genes. In some embodiments, the STRs of the target gene group include sequences of the loci D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, amelogenin gene, D5S818, and FGA. In some embodiments, the KIR genes in the target gene group include the following genes: 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, and 3DP1. It should be noted that the above-mentioned HLA, STR, and KIR gene fragments are highly polymorphic fragments in HLA, STR, and KIR, respectively, which reduces the probability (approximately 1 in 10 billion) of a gene completely identical to the target gene group in another sample during subsequent comparison, 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 test samples including a first test sample, a second test sample, and a third test sample.

[0038] In some embodiments, the first test sample comprises blood (used primarily 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 STRs. For example, human tissue cells include buccal cells, blood, hair, placenta, umbilical cord, amniotic fluid, saliva, or a combination thereof. In some embodiments, the third test sample comprises blood (used primarily 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 genes of the first test sample and the HLA genes of the target gene group, and obtains the comparison result of the HLA genes.

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

[0041] In some embodiments, the use of NGS can achieve high resolution of HLA up to 8 orders of magnitude, specifically, the first and second digits (first region) represent HLA serological typing or allelic genome, 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 substitutions), and the seventh and eighth digits (fourth region) represent alleles with base substitutions in the non-coding region.

[0042] That is, 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, compared with analyzing only the coding region, further analyzing and comparing the gene sequence of the non-coding region not only improves the accuracy of determining gene identity, but also incorporates the comparison results of the non-coding region into the reference, thereby improving the success rate of cell therapy or organ transplantation.

[0043] In some embodiments, step S130 includes comparing the gene identities of HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, or a combination thereof, where these subtypes are highly polymorphic subtypes. As can be appreciated, HLA genes are the most highly polymorphic category of human genes, and the accuracy of the gene comparison can be further improved by simultaneously selecting and comparing the highly polymorphic subtypes of HLA genes.

[0044] In some embodiments, the comparison result of the HLA genes indicates whether the HLA genes of the first test sample are identical to each HLA subtype of the target gene group. If the results of all the measured HLA subtypes are identical, the comparison result of the HLA genes is determined to be identical.

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

[0046] In some embodiments, step S140 includes extracting STRs from the 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 genes.

[0047] In some embodiments, step S140 includes comparing the genetic identity of loci D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, amelogenin gene, D5S818, FGA, or combinations thereof, which are highly polymorphic loci. As can be appreciated, STRs are a category of genes with high polymorphisms in human genes, and the accuracy of the genetic comparison can be improved by simultaneously selecting and comparing loci with high polymorphisms among 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 result of the KIR genes.

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

[0050] In some embodiments, to improve detection accuracy, multiple analytical methods can be combined and compared. For example, PCR-SSOP and PCR-SSP, which take a short time, can be performed first, and the results of the two test methods can be compared to see if they are the same, thereby reducing the errors present in each individual method. In another example, when it is difficult to design primers for a gene fragment, PCR-SSOP can be selected in combination with SBT, which has high sequence resolution, to compare the results of the two test methods to see if they are the same. This can save the time required to design primers when using PCR-SSP, and the limitations on gene detection segments that exist due to the difficulty of primer design.

[0051] In some embodiments, step S150 includes comparing the gene identities of 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, or combinations thereof, where these gene segments are highly polymorphic genes. As can be seen, KIR genes are a category of highly polymorphic genes in human genes and are highly associated with immune responses. By simultaneously selecting and comparing gene segments with high polymorphisms among KIR genes, the accuracy of the gene comparison can be improved and the risk of immune rejection during treatment can be reduced.

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

[0053] Step S220 compares the identity of HLA genes between the test sample and the target sample and obtains the comparison result of HLA genes. Step S230 compares the identity of STRs between the test sample and the target sample and obtains the comparison result of STRs. Step S240 compares the identity of KIR genes between the test sample and the target sample and obtains the comparison result of KIR genes. Step S250 determines that the test sample and the target sample are derived from the same individual if the comparison results of HLA, STR, and KIR genes are all identical. Step S220 substantially corresponds to step S130, step S230 substantially corresponds to step S140, step S240 substantially corresponds to step S150, and step S250 substantially corresponds to step S160. The analysis and comparison methods can be referred to above and will not be described again here.

[0054] As described above, method 200 analyzes and compares three highly polymorphic gene types (HLA genes, STR genes, and KIR genes), thereby reducing the probability of identical gene comparison results to 1 in 10 billion and improving the accuracy of interpretation. Therefore, if the gene comparison results are identical, it can be determined that the test sample and target sample are derived from the same individual, and can be used for subsequent treatment.

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

[0056] The following are examples of three types of genetic identity determination processes. Since the genes of the target sample and the test sample can be analyzed in the same manner, for simplicity, the following will all be referred to as the genetic analysis process of the test sample. It should be noted that the analysis of the HLA genes, 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] HLA genes are the most polymorphic system in humans, and the probability of HLA genes being completely identical is extremely low (depending on race, the probability of two individuals' HLA genes being completely identical ranges from 1 in 2 billion to 1 in 200,000). Therefore, by selecting HLA genes and comparing the genetic identity, the accuracy of determining the genetic identity between the target sample and the test sample can be improved. The analytical process is exemplified below.

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

[0060] Next, human leukocyte antigen (HLA) genes were extracted and amplified from the test samples using TBG Diagnostics' HLAssure SE human leukocyte antigen (SBT) genotyping reagents (HLAssure SE A Locus SBT Kit, Product No. 50110; HLAssure SE B Locus SBT Kit, Product No. 50210; HLAssure SE C Locus SBT Kit, Product No. 50410; HLAssure SE DRB1-EX Locus SBT Kit, Product No. 50350; HLAssure SE DQB1 Locus SBT Kit, Product No. 50510). The human leukocyte antigen (HLA) genes were then extracted and amplified using an HLA high-resolution typing sequencer (Applied Biosystems). TM The NGS method was used to analyze the sequences of HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 using a DNA analyzer (product number A41046, name: 3730xl DNA Analyzer). The sequencing resolution reached eight orders of magnitude, and the genotypes of both coding and non-coding regions were included.

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

[0062] It should be noted that the resolution of NGS is reflected by whether the gene sequence can be completely read. Taking HLA-A as an example, A01 can be subdivided into A01:01 (GGATCATC), A01:02 (GGATGAAG), A01:03 (GGATCTAG), etc. based on sequence differences. When using low-resolution analysis, for example, up to two digits, only the first four gene sequences, such as A01 (GGATXXXX), A02 (CTGGXXXX), and A03 (ATCGXXXX), can be compared, and the last four sequences are ignored, allowing for rapid detection of positive or negative results. However, using low-resolution analysis increases the likelihood of rejection or death in cell therapy or organ transplantation, even if HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ are all matched.

[0063] Therefore, compared to sequencing with a resolution of 2 or 4 digits (as is commonly known, when detection is performed up to 4 digits, comparisons that are 80% or more identical are judged to be from the same individual), the sequence resolution of the NGS of the present invention is improved to 8 digits (e.g., reading HLA-A A*01:01:01:01), and the genes must be completely identical, thereby improving the accuracy of reading the identity of HLA sequences and reducing the risk of rejection of subsequent treatment.

[0064] 2. STR identity

[0065] The genetic site of an STR is 3 to 7 base pairs in length, and STRs are widely distributed throughout the human genome and exhibit high levels of diversity. Therefore, by selecting STRs and performing identity comparisons, the accuracy of determining genetic identity between a target sample and a test sample can be improved. The analytical process is exemplified below.

[0066] First, human tissue cells (for example, buccal cells or blood) were provided as test samples.

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

[0068] Amplification kit (Brand: Applied Biosystems, Name: AmpFLSTR (R) Identifiler (R) The extracted STRs were amplified using a 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 the 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, the target sample and the test sample were compared to determine whether the genes at the above loci of the STR were completely identical, and the STR comparison results were obtained.

[0071] It should be noted that compared to selecting other loci of STRs, the combination of STR loci of the present invention can improve the accuracy rate of gene identity interpretation to 99% by selecting sites with high genetic diversity in STRs.

[0072] 3. KIR identity

[0073] KIR genes are a collective term for a group of genes located on human chromosome 19. The presence of KIR genes allows KIR proteins to be expressed in natural killer cells, which play an important role in human immune responses. There are a total of 17 KIR genes, but not all individuals express all KIR genes. Different combinations of KIR genes can combine different KIR gene subunits, resulting in a high degree of genetic polymorphism. Furthermore, in organ transplantation therapy, if the HLA genes are identical, the recipient and donor have the same KIR gene subunits, which can further improve the transplant success rate and reduce the risk of acute myeloid leukemia (AML). Therefore, having identical KIR genes (derived from the same individual) contributes to improving the success rate of subsequent cell therapy or organ transplantation.

[0074] The analysis process is illustrated below.

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

[0076] Next, we used the KIR typing kit (name: Exprobe) from TBG Biotechnology Corp. TM Using a KIR Typing Kit (product number: 69010) and a PCR machine (brand: Applied Biosystems, name: 7500 Real-Time PCR system), PCR was performed using sequence-specific primers for 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, and 3DP1 in KIR by the PCR-SSP method to confirm whether the gene fragment to be detected was present in the test sample.

[0077] As can be appreciated, any method that can be used to analyze the KIR genotypes described above can be used in the examples, including, but not limited to, PCR-SSP, PCR-SSOP, and SBT. If necessary, multiple sequencing methods can be combined simultaneously to improve detection accuracy.

[0078] Next, the distribution of the KIR gene block (sequence determination results in the SBT method) in the target sample and the test sample was compared to determine whether they were identical, and the results of the comparison of the KIR genes were obtained.

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

[0080] By comparing the three highly polymorphic gene sequences of Example 1 (HLA), Example 2 (STR), and Example 3 (KIR), the probability that the target gene group is identical to the test gene group can be reduced to approximately 1 in 10 billion. Therefore, if the three comparison results of HLA, STR, and KIR between the target sample and the test sample are all identical, theoretically, only one person in the global population will match, which is like locking three genes between the samples. Since the probability of genetic identity is extremely low, if the genes are identical, it is guaranteed that 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 of the test sample is identical to the target gene group of the target sample, thereby matching the individuals from which the test sample and the target sample are derived, and the test sample can be used for subsequent treatment.

[0082] The present disclosure has been disclosed in the above embodiments, but these are not intended to limit the present disclosure. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the scope of protection of the present disclosure is based on that defined 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. 1. A method for comparing genetic identity, comprising: Providing a group of target genes; providing test samples including a first test sample, a second test sample, and a third test sample; comparing the identity between the human leukocyte antigen genes of the first test sample and the human leukocyte antigen genes of the target gene group, and obtaining the comparison result of the human leukocyte antigen genes; comparing the identity between the short tandem repeat sequences 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; 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 result of the killer cell immunoglobulin-like receptor gene; determining that the target gene group is identical to the test gene group of the test sample when the comparison results of the human leukocyte antigen genes, the short tandem repeat sequence, and the killer cell immunoglobulin-like receptor gene are all identical; A method for comparing genetic identity, including:

2. The method of claim 1 , wherein the first test sample comprises 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 includes: extracting and amplifying the human leukocyte antigen gene from the first test sample; analyzing and comparing the identity of the human leukocyte antigen gene of the first test sample with the human leukocyte antigen gene in the target gene group using a next-generation sequencing method; The method of claim 1 , comprising:

4. The method of claim 1, wherein the step of comparing the identity between the human leukocyte antigen genes of the first test sample and the human leukocyte antigen genes of the target gene group comprises comparing the identity of genes for HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, or a combination 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 includes: comparing the identity of a coding region in the human leukocyte antigen gene of the first test sample with a coding region in the human leukocyte antigen gene of the target gene group; comparing the identity of a non-coding region in the human leukocyte antigen gene of the first test sample with that of a non-coding region in the human leukocyte antigen gene of the target gene group; The method of claim 1 , comprising:

6. The method of 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 comprises: extracting the short tandem repeat sequence from the second test sample; analyzing and comparing the identity of the short tandem repeat sequences of the second test sample with the short tandem repeat sequences in the target gene group using a sequence typing method; The method of claim 1 , comprising:

8. 2. The method of claim 1, wherein 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 comprises comparing the identity of genes at the following loci: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, amelogenin gene, D5S818, FGA, or a combination thereof.

9. The method of claim 1 , wherein the third test sample comprises 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 includes: extracting the killer cell immunoglobulin-like receptor gene from the third test sample; analyzing and 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 typing method by sequence, or a combination thereof; The method of claim 1 , comprising:

11. 2. The method of 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 comprises comparing the identity of genes 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, or a combination thereof.

12. 1. A method for determining the identity of individuals from which multiple samples are derived, comprising: providing a test sample and a target sample; comparing the identity of the human leukocyte antigen gene between the test sample and the target sample and obtaining a comparison result of the human leukocyte antigen gene; comparing the identity of the short tandem repeat sequences between the test sample and the target sample and obtaining a 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 a comparison result of the killer cell immunoglobulin-like receptor genes; determining that the test sample and the target sample are derived from the same individual when the results of the comparison of the human leukocyte antigen gene, the comparison of the short tandem repeat sequence, and the comparison of the killer cell immunoglobulin-like receptor gene are all identical; A method for determining the identity of an individual from which a plurality of samples are derived, comprising:

13. The method of claim 12 , wherein the test sample and the target sample comprise blood.

14. The step of comparing the identity of the human leukocyte antigen gene between the test sample and the target sample includes: extracting and amplifying the human leukocyte antigen gene from the test sample and the human leukocyte antigen gene from the target sample; analyzing and comparing the identity of the human leukocyte antigen gene of the test sample with the human leukocyte antigen gene of the target sample using next generation sequencing; 13. The method of claim 12, comprising:

15. 13. The method of claim 12, wherein comparing the identity of the human leukocyte antigen genes between the test sample and the target sample comprises comparing the identity of genes for HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, or a combination thereof.

16. The step of comparing the identity of the human leukocyte antigen gene between the test sample and the target sample includes: comparing the identity of a coding region in the human leukocyte antigen gene of the test sample with that of a coding region in the human leukocyte antigen gene of the target sample; comparing the identity of a non-coding region in the human leukocyte antigen gene of the test sample with that of a non-coding region in the human leukocyte antigen gene of the target sample; 13. The method of claim 12, comprising:

17. The step of comparing the identity of the short tandem repeat sequences between the test sample and the target sample comprises: extracting short tandem repeat sequences from the test sample and the target sample; analyzing and comparing the identity of the short tandem repeat sequences of the test sample with the short tandem repeat sequences of the target sample using a sequence-based typing method; 13. The method of claim 12, comprising:

18. 13. The method of claim 12, wherein comparing the identity of the short tandem repeat sequences between the test sample and the target sample comprises comparing the identity of genes at the following loci: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, 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 includes: extracting killer cell immunoglobulin-like receptor genes from the test sample and killer cell immunoglobulin-like receptor genes from the target sample; analyzing and comparing the identity of the killer cell immunoglobulin-like receptor gene of the test sample with that of the target sample using a sequence-specific oligonucleotide probe method, a polymerase chain reaction-sequence-specific primer method, a sequence typing method, or a combination thereof; 13. The method of claim 12, comprising:

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

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