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技術(shù)服務(wù)細(xì)胞鑒定

細(xì)胞鑒定

Cell identification

細(xì)胞鑒定

近年來，大量研究表明 STR 基因分型方法是進(jìn)行細(xì)胞交叉污染和性質(zhì)鑒定的最有效和準(zhǔn)確的方法之一，STR基因分型應(yīng)用于細(xì)胞鑒定已被ATCC等機(jī)構(gòu)強(qiáng)烈推薦。美國的ATCC 細(xì)胞庫、德國的DSMZ細(xì)胞庫以及日本的 JCRB細(xì)胞庫等為STR 分型提供了各細(xì)胞株的數(shù)據(jù)供比對(duì)。

STR基因位點(diǎn)由長度為2～7個(gè)堿基對(duì)的短串連重復(fù)序列組成，這些重復(fù)序列廣泛存在于人類基因組中，可作為高度多態(tài)性標(biāo)記，被稱為細(xì)胞的DNA指紋，其可通過PCR（聚合酶鏈?zhǔn)椒磻?yīng)）來檢測(cè)。STR基因座位上的等位基因可通過擴(kuò)增區(qū)域內(nèi)重復(fù)序列的拷貝數(shù)的不同來區(qū)分，在毛細(xì)管電泳分離之后可通過熒光檢測(cè)來識(shí)別。隨后通過一定的計(jì)算方法，即可根據(jù)所得的STR分型結(jié)果與專業(yè)的細(xì)胞STR數(shù)據(jù)庫比對(duì)從而推算出樣品所屬的細(xì)胞系或可能的交叉污染的細(xì)胞系名稱。

實(shí)驗(yàn)方案：

收樣
細(xì)胞DNA
提取
DNA擴(kuò)增
STR位點(diǎn)和
性別基因檢測(cè)
STR分型圖譜
和數(shù)據(jù)庫比對(duì)

樣本要求：

活細(xì)胞/細(xì)胞團(tuán)/凍存株數(shù)量要求：≥10^6個(gè)；
DNA送樣標(biāo)準(zhǔn)是：DNA濃度>50ng/μl，體積≥20μl；
活細(xì)胞一般為20℃以上常溫寄送，細(xì)胞團(tuán)藍(lán)冰寄送，DNA一般也是藍(lán)冰寄送，除非客戶特殊要求干冰出庫，凍存株使用干冰寄送。

報(bào)告樣例：

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參考文獻(xiàn)：

1. Chatterjee, R. (2007) Cell biology. Cases of mistaken identity. Science 315, 928-31.
2. Ruiz Bravo, N. and Gottesman, M. (2007) Notice regarding authentication of cultured cell lines. This can be viewed online at: http://grants.nih.gov/grants/guide/notice-files/NOT-OD-08-017.html
3. Yoshino, K. et al. (2006) Essential role for gene profiling analysis in the authentication of human cell lines. Human Cell 19, 43–8.
4. Szibor, R. et al. (2003) Cell line DNA typing in forensic genetics—the necessity of reliable standards. Forensic Sci. Int. 138, 37–43.
5. Dirks, W.G. et al. (2005) Short tandem repeat DNA typing provides an international reference standard for authentication of human cell lines. ALTEX 22, 103–9.
6. Masters, J.R. et al. (2001) Short tandem repeat profiling provides an international reference standard for human cell lines. Proc. Natl. Acad. Sci. USA 98, 8012–7.
7. (2001) Verify cell line identity with DNA profiling. ATCC Connection: Newsletter of The American Type Culture Collection 21, 1–2.
8. Krenke, B. et al. (2002) Validation of a 16-locus fluorescent multiplex system. J. Forensic Sci. 47, 773–85.
9. Levinson, G. and Gutman, G.A. (1987) Slipped-strand mispairing: A major mechanism for DNA sequence evolution. Mol. Biol. Evol. 4, 203–21.
10. Schlotterer, C. and Tautz, D. (1992) Slippage synthesis of simple sequence DNA. Nucleic Acids Res. 20, 211–5.
11. Smith, J.R. et al. (1995) Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase. Genome Res. 5, 312–7.
12. Magnuson, V.L. et al. (1996) Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: Implications for PCR-based genotyping. BioTechniques 21, 700–9.
13. Walsh, P.S., Fildes, N.J. and Reynolds, R. (1996) Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA. Nucleic Acids Res. 24, 2807–12.
14. Moller, A., Meyer, E. and Brinkmann, B. (1994) Different types of structural variation in STRs: HumFES/FPS, HumVWA and HumD21S11. Int. J. Leg. Med. 106, 319–23.
15. Brinkmann, B., Moller A. and Wiegand, P. (1995) Structure of new mutations in 2 STR systems. Int. J. Leg. Med. 107, 201–3.
16. Griffiths, R. et al. (1998) New reference allelic ladders to improve allelic designation in a multiplex STR system. Int. J. Legal Med. 111, 267–72.
17. Bär, W. et al. (1997) DNA recommendations: Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems. Int. J. Legal Med. 110, 175–6.
18. Gill, P. et al. (1997) Considerations from the European DNA Profiling Group (EDNAP) concerning STR nomenclature. Forensic Sci. Int. 87, 185–92.