Str methods for identifying human cells

Abstract

The present application belongs to the technical field of cell biology and bioinformatics, and provides a STR method for identifying human cells, which comprises the following steps: step 1, extracting cell DNA; step 2, diluting DNA; step 3, analyzing results; and step 4, checking frequency. The present application uses a new 21-site STR typing technology to detect cross contamination and identify the identity of human cell lines, and a total of 3 cell lines are detected to test the feasibility of the human cell STR method. The detection result shows that the detection result is consistent with the type of the expected target cell line.

Description

Technical Field

[0001] This invention belongs to the fields of cell biology and bioinformatics, specifically relating to the STR method for identifying human cells. Background Technology

[0002] Cell lines are crucial materials in life sciences and clinical medicine research, but the issue of cell quality identification has been neglected by many researchers. Cell contamination and misidentification are common. In recent years, many authoritative journals and research institutions have recommended that cell line identification be performed before publishing research papers or applying for grants. Although many new methods exist for cell identification, DNA typing based on short tandem repeats remains the gold standard. Short tandem repeats (STRs) are DNA sequences formed by tandem repeats of 2-7 base pairs as the core unit. Due to their wide distribution, high information content, high polymorphism, adherence to Mendelian inheritance laws, ease of PCR amplification and typing, STR technology has been widely used for paternity testing, detection of cross-contamination in human cell lines, and identification.

[0003] Advantages of STR genotyping: STR genotyping technology has 10 advantages: 1. The amplified DNA sequence is relatively short, approximately 100-350 bp. 2. Up to 20 sites can be amplified simultaneously in a single PCR reaction tube. 3. The entire process can be automated. 4. Technological advancements allow for the differentiation of single-base differential nucleotide fragments. 5. Commercially available kits and software are widely used and inexpensive. Results are reproducible in various laboratories and research institutions. 6. Many forensic laboratories offer economical and rapid STR genotyping services. 7. STR genotyping can be performed with as little as 2 ng of DNA. 8. STR sites are highly polymorphic. 9. STR sites are not linked across chromosomes. 10. Resolution increases with the number of STR sites.

[0004] Disadvantages of STR genotyping: The STR method has limitations in identifying human cells. Although primer pairs for other species, such as dogs and monkeys, have been developed, most primers used for cell identification can only detect human DNA. More importantly, some cell lines undergo mutations during passage due to allelic instability, leading to inaccurate STR detection. Many cancer cell lines are prone to STR loci changes, including allelic deletions and high-frequency microsatellite instability. Genetic drift can accumulate under in vitro subculture conditions. Excessive passage and dilution lead to subclonal selection. Conversely, subcloning can be reduced by selecting lower passage numbers and avoiding excessive dilution. For example, under favorable culture conditions, acute leukemia cell lines and CCRF-CEM cell lines exhibit considerable rapid passage drift in subclones, making STR identification inaccurate. In such cases, SNP genotyping should be considered for identifying genetically unstable cell lines. Single nucleotide polymorphisms (SNPs) refer to changes in genomic DNA at a specific nucleotide position, such as transitions, transversions, insertions, or deletions, with each allele having a frequency of at least 1% in the population. SNPs typically have only two alleles, i.e., biallelic mutations. Therefore, when detecting known mutation sites, only confirmatory analysis of the two different types of bases at the SNP site is needed, without requiring full-length DNA sequencing. Technically, any method that detects point mutations can be used to identify SNP mutations. Allele-specific hybridization has been used for high-throughput SNP identification, such as in the design and use of DNA microarrays. When referring to microarrays related to SNP sites, we are referring to microarrays that handle genotyping analysis, and the core of all genotyping microarrays is allele-specific oligonucleotides whose sequences are specific to a single allele. SNP technology is now used in forensic identification and cell identification. A 52-locus SNP genotyping technique has resolution comparable to a 16-locus STR technique, and so on. Similar to STR (Stereotyping and Generating) techniques, kits for SNP (Synthetic Nucleotide) techniques are also very common, such as the iPLEX Pro Sample ID Panel system. However, as a method for cell identification, SNP is not as widespread as STR because there is no sufficiently large reference database. SNP technology for cell line identification also has some advantages and disadvantages. Although SNP requires specific equipment, its advantages such as short typing time, low cost per sample, and readily available data for analysis continue to attract research institutions.

[0005] This invention utilizes a novel 21-locus STR typing technique for detecting and identifying cross-contamination in human cell lines. Three cell lines were tested to assess the feasibility of the human cell STR method. The results showed that the identified cell lines were consistent with the expected target cell line classification. Summary of the Invention

[0006] The purpose of this invention is to provide an STR method for identifying human cells, which can be used to identify cross-contamination between human cells or the origin of individual cells.

[0007] The objective of this invention can be achieved through the following technical solutions: The STR method for identifying human cells includes the following steps: Step 1: Cell DNA Extraction 1.1 Add 125 μL TL lysis buffer, 20 μL proteinase K, and 4 μL RNase to the cell samples submitted for testing, and digest in a shaker at 55℃ / 220 rpm for 1 h; 1.2 Cell genome extraction was performed using a nucleic acid extractor; Step 2, dilute the DNA 2.1 Determine the ng value of the genome after digestion and extraction of the cell stock solution; 2.2 Take 5 μL of the extracted genomic DNA from the cell stock solution and transfer it to a new EP tube. Add DEPC water to a concentration of 3 ng / μL and vortex to mix. 2.3 Prepare the reaction system on ice to reduce nonspecificity. After each reagent is added, check to ensure it has been added to the system. 2.4 Centrifuge the mixed system and place it in a PCR instrument; 2.5 Sample preparation for 3730; After preparing the reaction in a new 96-well half-skirt plate according to the above system, vortex and centrifuge, denature at 95°C for 3 min on a PCR instrument, quickly place it in a -20°C freezer for 3 min, and perform electrophoresis on a 3730xL sequencer. Step 3, Analysis Results The 3730xL sequencing file was analyzed using GeneMapper 5.0 software; Step 4: Review Frequency The document review frequency is set at once every six months to ensure that the documents are up-to-date and executable.

[0008] Furthermore, the plate-mounted reagents used in step 1.2 are as follows: The composition of deep well plate 1 is 200 μL binding solution + 400 μL anhydrous ethanol + 100 μL sample digestion solution; The composition of deep hole plate 2 is 80μL DEPC water + 20μL magnetic beads; The composition of deep well plate 3 is 600 μL of rinsing solution IR; The composition of deep well plate 4 is 600 μL of rinsing solution WB; The deep-hole plate 5 consists of 600 μL of rinsing solution WB; The composition of deep well plate 6 is 100 μL of elution buffer TE.

[0009] Furthermore, the reaction system in step 2.3 is as follows: PCR master Mix: 5 μL, STRtyper-21G Primer Mix (Plus): 2.5 μL, template: 1 μL, template: 1.5 μL, total: 10 μL.

[0010] Furthermore, the PCR instrument parameters in step 2.4 are as follows: 95℃: 5min, 1cycle; 94℃: 10s, 61℃: 60s, 70℃: 30s, 30cycle; 60℃: 15min, 1cycle; 4℃: ∞, 1cycle.

[0011] Furthermore, the preparation system for sample 3730 in step 2.5 is as follows: HiDi: 8.5μL, SiZE-500: 0.5μL, product: 1μL, total: 10μL.

[0012] The beneficial effects of this invention are: Compared to software analysis, the internal standard was not misaligned, and there were 1-3 peaks at 21 sites.

[0013] This invention is the first to use the new STR technique with 21 loci to identify and study three human cell lines.

[0014] This invention can accurately identify human cell types and distinguish between cell contamination in the shortest possible time.

[0015] This invention utilizes a novel 21-locus STR typing technique for detecting and identifying cross-contamination in human cell lines. Three cell lines were tested to assess the feasibility of the human cell STR method. The results showed that the identified cell lines were consistent with the expected target cell line classification. Attached Figure Description

[0016] The invention will now be further described with reference to the accompanying drawings.

[0017] Figure 1 This is the STR typing map of cell line 1 to be tested in this invention; Figure 2 This is the STR typing map of cell line 2 to be tested in this invention; Figure 3 This is the STR typing map of cell line 3 to be tested in this invention. Detailed Implementation

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1 This embodiment provides the STR method for identifying human cells, and the specific steps are as follows: Step 1: Cell DNA Extraction 1.1 Add 125 μL TL lysis buffer, 20 μL proteinase K, and 4 μL RNase to the cell samples submitted for testing, and digest in a shaker at 55℃ / 220 rpm for 1 h; 1.2 Cell genome extraction was performed using a nucleic acid extractor. The plate-mount reagents used are shown in Table 1. Table 1

[0020] Step 2, dilute the DNA 2.1 Determine the ng value of the genome after digestion and extraction of the cell stock solution; 2.2 Take 5 μL of the extracted genomic DNA from the cell stock solution and transfer it to a new EP tube. Add DEPC water to a concentration of 3 ng / μL and vortex to mix. 2.3 Prepare the reaction system on ice to reduce nonspecificity. After each reagent is added, check to ensure it has been added to the system. The reaction system is shown in Table 2. Table 2

[0021] 2.4 Centrifuge the mixed system and place it in a PCR instrument. The PCR instrument parameters are shown in Table 3. Table 3

[0022] 2.5 Sample preparation for 3730 is shown in Table 4: Table 4

[0023] After preparing the reaction in a new 96-well half-skirt plate according to the above system, vortex and centrifuge, denature at 95°C for 3 min on a PCR instrument, quickly place it in a -20°C freezer for 3 min, and perform electrophoresis on a 3730xL sequencer. Step 3, Analysis Results The 3730xL sequencing file was analyzed using GeneMapper 5.0 software; Step 4: Review Frequency The document review frequency is set at once every six months to ensure that the documents are up-to-date and executable.

[0024] I. Verification and Testing: To verify the correctness and reproducibility of the 21-site STR amplification system, three cell lines were selected for cellular STR experiments. The relevant information of the three cell lines is shown in Table 5. Table 5

[0025] II. Results and Analysis: Cell line 1: DNA typing of this cell line yielded a perfectly matching cell line in the cell line search. The EXPASY database shows the cell name as TE-15, with the cell number corresponding to CVCL_1763. No multiple alleles were detected in this cell line during this test.

[0026] Cell line 2: DNA typing of this cell line yielded a perfectly matching cell line in the cell line search. The EXPASY database shows the cell name as Huh-7, with cell number CVCL_0336 Best. No multiple alleles were detected in this cell line.

[0027] Cell line 3: DNA typing of this cell line yielded a perfectly matching cell line in the cell line search. The EXPASY database shows the cell name as Hep 3B2.1-7, with the cell number CVCL_0326 Best. No multiple alleles were detected in this cell line.

[0028] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0029] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The STR method for identifying human cells, characterized by comprising the following steps: Step 1: Cell DNA Extraction 1.1 Add 125 μL TL lysis buffer, 20 μL proteinase K, and 4 μL RNase to the cell samples submitted for testing, and digest in a shaker at 55℃ / 220 rpm for 1 h; 1.2 Cell genome extraction was performed using a nucleic acid extractor; Step 2, dilute the DNA 2.1 Determine the ng value of the genome after digestion and extraction of the cell stock solution; 2.2 Take 5 μL of the extracted genomic DNA from the cell stock solution and transfer it to a new EP tube. Add DEPC water to a concentration of 3 ng / μL and vortex to mix. 2.3 Prepare the reaction system on ice. After each reagent is added, check to ensure it has been added to the system. 2.4 Centrifuge the mixed system and place it in a PCR instrument; 2.5 Sample preparation for 3730; After preparing the reaction mixture in a new 96-well half-skirt plate according to the above system, centrifuge with shaking, denature at 95°C for 3 min on a PCR instrument, cool rapidly at -20°C for 3 min, and then perform electrophoresis. Step 3: Analyze the results; Step 4: Review the frequency.

2. The STR method for identifying human cells according to claim 1, characterized in that the plate-position reagent used in step 1.2 is as follows: The composition of deep well plate 1 is 200 μL binding solution + 400 μL anhydrous ethanol + 100 μL sample digestion solution; The composition of deep hole plate 2 is 80μL DEPC water + 20μL magnetic beads; The composition of deep well plate 3 is 600 μL of rinsing solution IR; The composition of deep well plate 4 is 600 μL of rinsing solution WB; The deep-hole plate 5 consists of 600 μL of rinsing solution WB; The composition of deep well plate 6 is 100 μL of elution buffer TE.

3. The STR method for identifying human cells according to claim 1, characterized in that, The reaction system in step 2.3 is as follows: PCR master Mix: 5 μL, STRtyper-21G Primer Mix (Plus): 2.5 μL, template: 1 μL, template: 1.5 μL, total: 10 μL.

4. The STR method for identifying human cells according to claim 1, characterized in that, The PCR instrument parameters in step 2.4 are as follows: 95℃: 5min, 1cycle; 94℃: 10s, 61℃: 60s, 70℃: 30s, 30cycle; 60℃: 15min, 1cycle; 4℃: ∞, 1cycle.

5. The STR method for identifying human cells according to claim 1, characterized in that, The preparation system for sample 3730 in step 2.5 is as follows: HiDi: 8.5μL, SiZE-500: 0.5μL, product: 1μL, total: 10μL.

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