Ampflstr identifiler plus pcr amplification kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The AmpFlSTR® Identifiler® Plus PCR Amplification Kit is a short tandem repeat (STR) multiplex assay designed for human identification. The kit amplifies 15 STR loci and the amelogenin gender determination marker in a single PCR reaction. The amplified fragments can be detected and analyzed using capillary electrophoresis instruments.

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32 protocols using ampflstr identifiler plus pcr amplification kit

Characterization of HPV-positive and HPV-null Cell Lines

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HeLa (HPV18 cervical cancer cells), CaSki (HPV16 cervical cancer cells), human embryonic kidney (HEK) 293 (HPV-null epithelial cells), C33A (HPV-null cervical cancer cells), U-2 OS (HPV-null human osteosarcorma cells), HeKa (HPV-null primary keratinocytes), SCC25 (HPV-null human head and neck head and neck squamous cell carcinoma [HNSCC] cell line), and SCC152 (HPV16-positive human HNSCC cell line) were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS, GIBCO) at 37°C in a humidified incubator with 5% CO₂. Identities of these human cell lines were validated by short tandem repeat (STR) profiling using the AmpFlSTR Identifiler Plus PCR Amplification Kit (Thermo Fisher Scientific) with the Applied Biosystems 3500 Series Genetic Analyzer, and analyzed by GeneMapper Software 5 (Applied Biosystems). The STR profile of all cells lines showed >88% concordance with their reference profiles in the ATCC cell line database.

Li S., Yim M.K., Yip K.L., Xiao C., Luk H.Y., Xiao S., Chen Z., Chan P.K, & Boon S.S. (2023). E6-Encoded by Cancer-Causing Human Papillomavirus Interacts with Aurora Kinase A To Promote HPV-Mediated Carcinogenesis. Journal of Virology, 97(2), e01872-22.

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STR Typing of Genomic Samples

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Conventional STR typing was performed on the 12 genomic samples using the AmpFlSTR® Identifiler® Plus PCR Amplification Kit (Thermo Fisher Scientific) and the AmpFlSTR® Yfiler® PCR Amplification Kit (Thermo Fisher Scientific) and one nanogram of DNA for each reaction per the recommended manufacturer’s protocols (13 ,14 ). The GeneAmp® PCR System 9700 thermal cycler (Thermo Fisher Scientific) was used for PCR amplification. Electrophoresis was completed on an ABI Prism® 3100xl Genetic Analyzer (Thermo Fisher Scientific). Raw data were analyzed with GeneMapper® ID software v3.2.1 (Thermo Fisher Scientific).

Churchill J.D., Chang J., Ge J., Rajagopalan N., Wootton S.C., Chang C.W., Lagacé R., Liao W., King J.L, & Budowle B. (2015). Blind study evaluation illustrates utility of the Ion PGM™ system for use in human identity DNA typing. Croatian Medical Journal, 56(3), 218-229.

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Human Cell Lines Maintenance Protocol

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HeLa (HPV18 cervical cancer cells), CaSki (HPV16 cervical cancer cells), Human embryonic kidney (HEK) 293 (HPV-null epithelial cells), C33A (HPV-null cervical cancer cells) and U-2 OS (HPV-null human osteosarcoma cells) were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS, GIBCO) at 37 °C in a humidified incubator with 5% CO₂. Human primary normal epidermal keratinocytes, adult (HeKa) cell line was purchased from ThermoFisher Scientific (Waltham, MA, USA) and maintained in EpiLife^TM medium supplemented with human keratinocyte growth supplement (HKGS). Identities of these human cell lines were validated by short tandem repeat (STR) profiling using the AmpFlSTR Identifiler^® Plus PCR Amplification Kit (ThermoFisher Scientific) with the Applied Biosystems 3500 Series Genetic Analyzer and analyzed by GeneMapper^® Software 5 (Applied Biosystems, Bedford, MA, USA). The STR profile of all cell lines showed >88% concordance with their reference profiles in the ATCC cell line database.

Boon S.S., Lee Y.C., Yip K.L., Luk H.Y., Xiao C., Yim M.K., Chen Z, & Chan P.K. (2023). Interaction between Human Papillomavirus-Encoded E6 Protein and AurB Induces Cell Immortalization and Proliferation—A Potential Target of Intervention. Cancers, 15(9), 2465.

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Cell Line Characterization and Validation

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Early passage MEFs (P3–P4) from E13.5 embryos were used for experiments. Mouse lung cancer (KLA-KR181^fl/fl) and pancreatic cancer (KPC181^wt and KPC181^ko) cell lines were isolated from corresponding GEMMs. MEFs and mouse cancer cell lines were grown in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Human non–small cell lung cancer cells used were either wild-type KRAS (NCI-H2126) or mutant KRAS (NCI-H1792), were grown in RPMI1640 supplemented with 10% FBS and 1% penicillin-streptomycin, and were acquired from ATCC. 3KT cells were a gift from John Minna (UTSouthwestern, Dallas, TX, USA) (79 (link)). 3KT and H6c7 cells (Kerafast Inc.) were grown in keratinocyte medium (GIBCO). Human cancer cell lines were authenticated by the Genomics Unit at Center for Applied Medical Research (CIMA) using Short Tandem Repeat profiling (AmpFLSTR Identifiler Plus PCR Amplification Kit, Thermo Fisher Scientific). Cell lines were tested with the MycoAlert Mycoplasma Detection Kit (LONZA). Only mycoplasma-negative cells were used.

Valencia K., Erice O., Kostyrko K., Hausmann S., Guruceaga E., Tathireddy A., Flores N.M., Sayles L.C., Lee A.G., Fragoso R., Sun T.Q., Vallejo A., Roman M., Entrialgo-Cadierno R., Migueliz I., Razquin N., Fortes P., Lecanda F., Lu J., Ponz-Sarvise M., Chen C.Z., Mazur P.K., Sweet-Cordero E.A, & Vicent S. (2020). The Mir181ab1 cluster promotes KRAS-driven oncogenesis and progression in lung and pancreas. The Journal of Clinical Investigation, 130(4), 1879-1895.

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Identity Testing Using AmpFlSTR Identifiler Plus

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For identity testing, we used the AmpFlSTR Identifiler Plus PCR Amplification Kit (Thermo Fisher Scientific, cat. no. 4427368) according to the manufacturer’s instructions. The kit uses a five-dye fluorescent system, and the inclusion of non-nucleotide linkers allows for simultaneous amplification and efficient separation of 15 STR loci. Electropherograms of the fragments were analyzed using NCBI’s OSIRIS 2.13.1 program (Table S1).

Morato Torres C.A., Zafar F., Tsai Y.C., Vazquez J.P., Gallagher M.D., McLaughlin I., Hong K., Lai J., Lee J., Chirino-Perez A., Romero-Molina A.O., Torres F., Fernandez-Ruiz J., Ashizawa T., Ziegle J., Jiménez Gil F.J, & Schüle B. (2022). ATTCT and ATTCC repeat expansions in the ATXN10 gene affect disease penetrance of spinocerebellar ataxia type 10. Human Genetics and Genomics Advances, 3(4), 100137.

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Cell Line Validation and Culture Conditions

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293T cells were grown from a master cell bank [67 (link)] and Jurkat (Clone E6-1) cells were obtained from ATCC. Both cell lines were maintained as lab frozen stocks and validated at the time of study by tandem repeat analysis using the Applied Biosystems AmpFLSTR^™ Identifiler^™ Plus PCR Amplification Kit (Thermo Fisher Scientific, Carlsbad, CA). Jurkat cells were cultured in RPMI supplemented with 10% FBS (Gemini), 100 U/mL penicillin, 100 μg/mL streptomycin, 2mM glutamine and 55μM β-mercaptoethanol at 1 x 10⁶ cell/ml, while Human Embryonic Kidney (HEK) 293 T cells were grown in DMEM supplemented with 10% FBS (Gemini) and 125 μM gentamycin. Both cell lines were maintained in a 37°C incubator containing 5% CO₂.

Read D.F., Atindaana E., Pyaram K., Yang F., Emery S., Cheong A., Nakama K.R., Burnett C., Larragoite E.T., Battivelli E., Verdin E., Planelles V., Chang C.H., Telesnitsky A, & Kidd J.M. (2019). Stable integrant-specific differences in bimodal HIV-1 expression patterns revealed by high-throughput analysis. PLoS Pathogens, 15(10), e1007903.

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Multiplex PCR and qPCR Analysis

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Multiplex PCR amplification of V01-V10 and aCh_1–13 samples, in addition to the patient samples pD2, pR2 and pCh_1–6, was performed according to the manufacturer's instructions of the AmpFlSTR Identifiler Plus PCR Amplification kit (Thermo Fisher Scientific, Inc.). Amplicons were resolved on a Genetic Analyzer 3130 and analyzed with GeneMapper software, version 4.1 (Life Technologies; Thermo Fisher Scientific, Inc.).
Patient samples (pCh_1–6) were also analyzed by qPCR (data not shown), as previously investigated by Bai et al (14 (link)). This analysis was performed as an additional validation method of NGS data, where a discrepancy between NGS and STR data was present.

Aloisio M., Licastro D., Caenazzo L., Torboli V., D'eustacchio A., Severini G.M, & Athanasakis E. (2016). A technical application of quantitative next generation sequencing for chimerism evaluation. Molecular Medicine Reports, 14(4), 2967-2974.

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Zygosity Determination via Genetic Profiling

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Zygosity testing (MZ versus DZ) was performed on blood DNA samples. Short tandem repeat polymorphic DNA markers were amplified by PCR using AmpFLSTR® Identifiler® Plus PCR Amplification Kit (Thermo Fisher Scientific, USA), labeled with fluorescent markers and separated by capillary electrophoresis to distinguish different alleles at each of 15 different loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TROX, D18S51, D5S818, and FGA).

Vilchez-Vargas R., Skieceviciene J., Lehr K., Varkalaite G., Thon C., Urba M., Morkūnas E., Kucinskas L., Bauraite K., Schanze D., Zenker M., Malfertheiner P., Kupcinskas J, & Link A. (2022). Gut microbial similarity in twins is driven by shared environment and aging. EBioMedicine, 79, 104011.

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Genetic Profiling of Gestational Trophoblastic Diseases

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To identify the genetic sources of GTDs, STR analysis was performed using genomic DNA from GTD tissues and matched normal cells using the AmpFLSTR Identifiler Plus PCR Amplification Kit (Thermo Fisher Scientific) that covered 16 STR loci (polymorphic DNA markers): D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, Amelogenin, D5S818, and FGA. The fluorescent PCR products were analyzed using the ABI 3130XL Genetic Analyzer (Applied Biosystems). Each fluorescent peak was quantified by its size (base pairs), peak height, and peak area, as previously reported^{15 (link)}, and analyzed by GeneMapper 4.1 software (Applied Biosystems).

Jung S.H., Choi Y.J., Kim M.S., Park H.C., Han M.R., Hur S.Y., Lee A.W., Shin O.R., Kim J., Lee S.H., Hong D., Song S.Y., Chung Y.J, & Lee S.H. (2020). Distinct genomic profiles of gestational choriocarcinoma, a unique cancer of pregnant tissues. Experimental & Molecular Medicine, 52(12), 2046-2054.

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Cell Line Characterization and Validation

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HEK293, HeLa, NIH/3T3 and U‐2 OS cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco's modified eagle medium, supplemented with 10% FBS in a 37°C humidified incubator containing 5% CO₂. Established human cell lines used in this study were short tandem repeat (STR) profiled using AmpFlSTR Identifiler^® Plus PCR Amplification Kit (ThermoFisher Scientific, Waltham, MA, USA) with the Applied Biosystems 3500 Series Genetic Analyzer and analysed by GeneMapper^® Software 5 (Applied Biosystems, Foster City, CA, USA). The STR profile of all cells lines showed >88% concordance with their reference profile in the ATCC cell line database.

Law P.T., Boon S.S., Hu C., Lung R.W., Cheung G.P., Ho W.C., Chen Z., Massimi P., Thomas M., Pim D., Banks L, & Chan P.K. (2018). Oncogenic comparison of human papillomavirus type 58 E7 variants. Journal of Cellular and Molecular Medicine, 23(2), 1517-1527.

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