Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ

Zischler, Hans; Nanda, Indrajit; Schäfer, Renate; Schmid, Michael; Epplen, Jörg T.

doi:10.1007/bf00291160

Cited by 100 publications

(27 citation statements)

References 9 publications

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“…9 The single-locus satellites are localized at specific sites on each human chromosome, while multi-locus satellite elements comprising short tandem repeats (STRs) are spread throughout the entire genome. 8,10 Polymorphic variation in the sizes of the DNA repeats -variable numbers of tandem repeats (VNTR) -define the length of the alleles, which may be distinguished electrophoretically by the Southern blot or polymerase chain reaction (PCR) techniques. The likelihood of individuals displaying distinct (paternal/maternal-derived) pairs of allelic DNA fragments depends directly on the degree of polymorphic variation encountered in their host genetic populations at that locus and on the frequency of the alleles in question.…”

Section: Dna Fingerprintingmentioning

confidence: 99%

False leukemia–lymphoma cell lines: an update on over 500 cell lines

et al. 2003

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Human leukemia-lymphoma (LL) cell lines represent an extremely important resource for research in a variety of fields and disciplines. As the cell lines are used as in vitro model systems in lieu of primary cell material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. Obviously, proper authentication of cell line derivation and precise characterization are indispensable requirements to use as model systems. A number of studies has shown an unacceptable level of LL cell lines to be false. We present here the results of authenticating a comprehensively large sample (n = 550) of LL cell lines mainly by DNA fingerprinting and cytogenetic evaluation. Surprisingly, nearidentical incidences (ca 15%) of false cell lines were observed among cell lines obtained directly from original investigators (59/395: 14.9%) and from secondary sources (23/155: 14.8%) implying that most cross-contamination is perpetrated by originators, presumably during establishment. By comparing our data with those published, we were further able to subclassify the false cell lines as (1) virtual: cross-contaminated with and unretrievably overgrown by other cell lines during initiation, never enjoying independent existence; (2) misidentified: crosscontaminated subsequent to establishment so that an original prototype may still exist; or (3) misclassified: unwittingly established from an unintended (often normal) cell type. Prolific classic leukemia cell lines were found to account for the majority of cross-contaminations, eg CCRF-CEM, HL-60, JUR-KAT, K-562 and U-937. We discuss the impact of cross-contaminations on scientific research, the reluctance of scientists to address the problem, and consider possible solutions. These findings provide a rationale for mandating the procurement of reputably sourced LL cell lines and their regular authentication thereafter.

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Section: Dna Fingerprintingmentioning

confidence: 99%

False leukemia–lymphoma cell lines: an update on over 500 cell lines

et al. 2003

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“…After hybridization overnight, essentially following ref. 26, slides were washed, with the highest stringency wash being at Tm -5°C. Probe detection, counterstaining, chromosome examination, image acquisition and processing was as described (22).…”

Section: Methodsmentioning

confidence: 99%

The physical and genomic organization of microsatellites in sugar beet.

Schmidt

Heslop-Harrison

1996

Proc. Natl. Acad. Sci. U.S.A.

100

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Microsatellites, tandem arrays of short (2-5 bp) nucleotide motifs, are present in high numbers in most eukaryotic genomes. We have characterized the physical distribution of microsatellites on chromosomes of sugar beet (Beta vulgaris L.). Each microsatellite sequence shows a characteristic genomic distribution and motif-dependent dispersion, with site-specific amplification on one to seven pairs of centromeres or intercalary chromosomal regions and weaker, dispersed hybridization along chromosomes. Exclusion of some microsatellites from 18S-5.8S-25S rRNA gene sites, centromeres, and intercalary sites was observed. In-gel and in situ hybridization patterns are correlated, with highly repeated restriction fragments indicating major centromeric sites ofmicrosatellite arrays. The results have implications for genome evolution and the suitability of particular microsatellite markers for genetic mapping and genome analysis.Runs of repetitions of short sequence motifs 2-5 bp long, described as microsatellites or simple sequence repeats, are probably ubiquitous elements of eukaryotic genomes (1-3). They provide highly informative and polymorphic markers for genetics (4-6) or plant, fungal, and animal fingerprinting (7,8). Genetic mapping using microsatellites as markers involves amplification of repeat arrays using PCR with primers flanking the arrays. Primers are often chosen based on database searches or sequence data from cloned arrays (9-12), whichgive selective data about genomic distribution. However, little is known about the real chromosomal organization and physical localization of microsatellite motifs within plant genomes. The only microsatellite repeat mapped physically in plants, the polypurine motif (GAA)7, has been correlated with the positions of C-bands in barley (13). Some physical mapping in fish and primates (8, 14) using in situ hybridization shows clustering of microsatellites on some chromosomes. Recent data in mouse show that mapped microsatellites, mostly (CA)n repeats mapped by PCR polymorphisms, are distributed among autosomes in proportion to chromosome length, while the X chromosome shows a clear deficit of the microsatellites (15). Within chromosomes, the frequency of both large and small clusters with respect to meiotic crossovers slightly exceeded expectation.Sugar beet (Beta vulgaris L.; 2n = 2x = 18) is a valuable model species for investigating the large scale organization of the nuclear genome because (i) the genome is relatively small with 758 Mbp (16), (ii) fluorescent in situ hybridization can accurately locate sequences along the metaphase chromosomes and within interphase nuclei (17), (iii) major classes of the repetitive DNA have been characterized including both satellite and retrotransposon sequences (18)(19)(20)(21)(22), and (iv) microsatellites are known to be highly abundant (23).Here, we aimed to characterize the genomic distribution of microsatellite sequences in sugar beet. We used seven simple sequences representing a range of nucleotide motifs, many used for g...

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“…in comparison to the cloned minisatellites probes). Nonradioactive, digoxigenated oligonucleotide probes with nearly equivalent sensitivity are also available [16].…”

Section: Discussionmentioning

confidence: 99%

“…In principle, simple repeats have similar properties to the minisatellites, yet they are spread ubiquitously over the human chromosomes, located in intergenic spacers and introns [15,16]. Both probes show extensive polymorphisms.…”

Section: Introductionmentioning

confidence: 99%

Determining consanguinity by oligonucleotide fingerprinting with (GTG)₅/(CAC)₅

et al. 1991

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Simple tandemly organized (GTG)n/(CAC)n sequences are spread throughout the human chromosomes. The most informative DNA fingerprints for the testing of pedigrees and/or paternity were obtained with the simple triplet repeat probe (GTG)5 or its complement (CAC)5. These hypervariable simple-repeat fragments are stably inherited in a Mendelian fashion. Using these highly discriminating probes, all human individuals could, theoretically, be differentiated, except for genetically identical monozygotic twins. Examples from actual case work are reported and pertinent advantages of this methodology are discussed.

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Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ

Cited by 100 publications

References 9 publications

False leukemia–lymphoma cell lines: an update on over 500 cell lines

False leukemia–lymphoma cell lines: an update on over 500 cell lines

The physical and genomic organization of microsatellites in sugar beet.

Determining consanguinity by oligonucleotide fingerprinting with (GTG)₅/(CAC)₅

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Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ

Cited by 100 publications

References 9 publications

False leukemia–lymphoma cell lines: an update on over 500 cell lines

False leukemia–lymphoma cell lines: an update on over 500 cell lines

The physical and genomic organization of microsatellites in sugar beet.

Determining consanguinity by oligonucleotide fingerprinting with (GTG)5/(CAC)5

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Product

Resources

About

Determining consanguinity by oligonucleotide fingerprinting with (GTG)₅/(CAC)₅