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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/3.1/1.3


TITLE: DNA FINGERPRINTING


Identity tests rely in general on the detection of genetic differences among individuals. Highly polymorphic loci, the alleles of which are the result of variable tandem repeat sequences (VNTRs), are the most informative genetic markers for DNA fingerprinting. Several loci of VNTR sequences suitable for authentication purposes have been identified and the distribution of alleles of the VNTR loci has been studied in different populations (1-3). At present, the genetic authentication methods comprise fingerprints using multilocus probes (1-3) as well as probes for highly polymorphic single loci (4, 5) which have all considerable individualization potential. Multilocus probes detect in general short tandem repeats (STRs) spread throughout the entire genome while the information obtained using single locus probes is restricted to a minute spot in the large genome with the advantage of detecting both alleles.

The analysis of the frequency distribution of alleles generates a cell line-specific DNA profile. For this purpose, a multiplex polymerase chain reaction (PCR) is performed using genomic DNA of the respective cell line. The amplification of VNTR sequences at the loci D2S44 (6), D1S80 (7), D17S30 (8) and of the 3´ non-translated region of the Apo B gene (9) results in 4 to 8 DNA fragments of different sizes (170-4400 bp) which are separated on agarose gels. Measurement and digitization of the signal pattern enable the storage of a specific DNA profile in a database; a unique computer program allows for the comparison of fingerprint profiles (10).

The searchable database is based on a high degree of polymorphism which is in total >99.9% of the analyzed loci. In case of signal identity, the respective cell lines are analyzed using the conventional Southern technique combined with a (gtg)5 multilocus probe (11). This procedure verifies cell line authenticity during the accessioning process and confirms cell line identity after replenishment of the distribution stock. Previously, only the multilocus probe (gtg)5 or the single locus probe pYNH24 had been used for DNA fingerprinting.

References:

1. Jeffreys AJ, Wilson V, Thein SL: Hypervariable 'minisatellite' regions in human DNA. Nature 314: 67-73 (1985).

2. Zischler H, Nanda I, Schäfer R, Schmid M, Epplen JT: Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ. Hum Genet 82: 227-233 (1989).

3. Häne B, Tümmler M, Jäger K, Schleithoff L, Janssen JWG, Drexler HG: Differences in DNA fingerprints of continuous leukemia-lymphoma cell lines from different sources. Leukemia 6: 1129-1133 (1992).

4. Jeffreys AJ, Wilson V, Thein SL: Individual-specific 'fingerprints' of human DNA. Nature 316: 76-79 (1985).

5. Gilbert DA, Reid YA, Gail MA, Pee D, White C, Hay RJ, O'Brien SJ: Application of DNA fingerprints for cell-line individualization. Am J Hum Genet 47: 499-514 (1990).

6. Nakamura Y, Leppert M, O´Connell P, Wolff R, Holm T, Culver M, Martin C, Fujimoto E, Hoff M, Kumlin E, White R: Variable number of tandem repeat (VNTR) markers for human gene mapping. Science 235: 1616-1622 (1987).

7. Budowle B, Chakraborty R, Giusti AM, Eisenberg AJ, Allen RC: Analysis of the VNTR locus D1S80 by the PCR followed by high-resolution PAGE. Am J Hum Genet 48: 137-144 (1991).

8. Wolff RK, Nakamura Y, White R: Molecular characterization of a spontaneously generated new allele at a VNTR locus: No exchange of flanking DNA sequence. Genomics 3: 347-351 (1988).

9. Boerwinkle E, Xiong WJ, Fourest E, Chan L: Rapid typing of tandemly repeated hypervariable loci by the polymerase chain reaction: Application to the apolipoprotein B 3´ hypervariable region. Proc Natl Acad Sci USA 86: 212-216 (1989).

10. MacLeod RAF, Dirks WG, Reid YA, Hay RJ, Drexler HG: Identity of original and late-passage Dami megakaryocytes with HEL erythroleukemia cells shown by combined cytogenetics and DNA fingerprinting. Leukemia 11: 2032-2038 (1997).

11. Dirks W, Jäger K, Drexler HG: Evaluation of fingerprint methods for verification of cell line authenticity. (in preparation).

 


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