Sources of variability in data from a <i>lacl</i> transgenic mouse mutation assay

Piegorsch, Walter W.; Lockhart, Ann-Marie; Margolin, Barry H.; Tindall, Kenneth R.; Gorelick, Nancy J.; Short, Jay M.; Carr, Gregory J.; Thompson, Elizabeth I.; Shelby, Michael D.

doi:10.1002/em.2850230105

Cited by 61 publications

(18 citation statements)

References 17 publications

(4 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The 370-fold variation in a mutation frequency by Bi-PAP-A is consistent with the reported other measurements by the BigBlue mutation detection system, which are compatible with variable amounts of clonal expansion from the original mutation (37,38). The clonal expansion is a major contributor to the hyper-Poisson variance of mutant frequencies observed in BigBlue and other systems (37,38).…”

Section: Discussionsupporting

confidence: 89%

Detection of extremely rare alleles by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification (Bi-PAP-A): measurement of mutation load in mammalian tissues

Liu

Sommer

2004

BioTechniques

View full text Add to dashboard Cite

Pyrophosphorolysis-activated polymerization (PAP) was developed to detect extremely rare mutations in complex genomes. In theory, PAP can detect a copy of a single base mutation present in 3 x 10(11) copies of the wild-type allele. In practice, the selectivity of detection is limited by a bypass reaction involving a polymerase extension error from the unblocked oligonucleotide annealed to the opposing strand. Bidirectional PAP allele-specific amplification (Bi-PAP-A) is a novel method that uses two opposing 3'-terminal blocked pyrophosphorolysis-activatable oligonucleotides (P*s) with one nucleotide overlap at their 3' termini. This eliminates the problematic bypass reaction. The selectivity of Bi-PAP-A was examined using lambda phage DNA as a model system. Bi-PAP-A selectively detected two copies of a rare mutated allele in the presence of at least 2 x 10(9) copies of the wild-type lambda phage DNA. Bi-PAP-A was then applied to direct detection of spontaneous somatic mutations in the mouse genome at a frequency as low as 3 x 10(-9). A 370-fold variation in the frequency of a specific somatic mutation among different mouse samples was found, suggesting clonal expansion of mutation occurring during early development and a hyper-Poisson variance. Bi-PAP-A is a rapid, general, and automatable method for the detection of rare mutations.

show abstract

Section: Discussionsupporting

confidence: 89%

Detection of extremely rare alleles by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification (Bi-PAP-A): measurement of mutation load in mammalian tissues

Liu

Sommer

2004

BioTechniques

View full text Add to dashboard Cite

show abstract

“…detection system, compatible with a variable amount of clonal expansion from the original mutation. The clonal expansion is a major contributor to the hyper-Poisson variance of mutant frequencies observed in BigBlue s and other systems [Piegorsch et al, 1994;Nishino et al, 1996].…”

Section: à7mentioning

confidence: 99%

PAP: Detection of ultra rare mutations depends on P* oligonucleotides: ?Sleeping Beauties? awakened by the kiss of pyrophosphorolysis

Liu

Sommer

2004

Hum. Mutat.

View full text Add to dashboard Cite

For the Mutation Detection 2003 Special IssuePyrophosphorolysis-activated polymerization (PAP) was initially developed to enhance the specificity of allelespecific PCR for detection of known mutations in the presence of a great excess of wild-type allele. The high specificity of PAP derives from the serial coupling of activation of a 3 0 blocked pyrophosphorolysis-activable oligonucleotide (P n ) with extension of the unblocked, activated P n . In theory, PAP can detect a copy of a single base mutation present in 3 Â 10 11 copies of the wild-type allele. In practice, the selectivity of detection is limited by polymerase extension errors, a bypass reaction, from the unblocked oligonucleotide annealed to the opposing strand. Bi-directional PAP allele-specific amplification (Bi-PAP-A) is a derivative of PAP that uses two opposing pyrophosphorolysis activable oligonucleotides (P n ) with one nucleotide overlap at their 3 0 termini. This eliminates the problematic bypass reaction. The selectivity of Bi-PAP-A was examined using k phage DNA as a model system. Bi-PAP-A selectively detected two copies of a rare mutated allele in the presence of at least 2 Â 10 9 copies of the wild-type k phage DNA. We then applied Bi-PAP-A to direct detection of spontaneous somatic mutations in the lacI transgene in BigBlue transgenic mice at a frequency as low as 3 Â 10 À9. A 370-fold variation in the frequency of a specific somatic mutation among different mouse samples was found, implying hyper-Poisson variance and clonal expansion of mutation occurring during early development. Bi-PAP-A is a simple, rapid, and general method capable of automation and particularly suited to detection of ultra rare mutations. We also show that P n oligonucleotides have the novel and unexpected property of high specificity to mismatches with the template throughout lengths of the P

show abstract

“…Data were examined as described by Piegorsch et al (6). Because no excess variability was found between packaging reactions and packaging dates, subsequent analyses used pooled data.…”

Section: Animalsmentioning

confidence: 99%

Mutation frequency declines during spermatogenesis in young mice but increases in old mice

Walter

Intano

McCarrey

et al. 1998

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

125

121

View full text Add to dashboard Cite

Five percent of live-born human offspring will have a genetic disorder. Of these, 20% are because of germ-line de novo mutations. Several genetic diseases, such as neurofibromatosis and Duchenne muscular dystrophy, are associated with a high percentage of de novo germ-line mutations. Until recently, a direct analysis of spontaneous mutation frequencies in mammalian germ cells has been prevented by technical limitations. We have measured spontaneous mutation frequencies in a lacI transgene by using enriched populations of specific spermatogenic cell types. Similar to previously published results, we observed a lower mutation frequency for seminiferous tubule cell preparations, which contain all stages of spermatogenesis, relative to somatic tissues. We made the unexpected observation of a decline in mutation frequency during spermatogenesis, such that the mutation frequencies of type B spermatogonia and all subsequent stages of spermatogenesis are lower than the frequency for primitive type A spermatogonia. In addition, spermatogenic cells from old mice have significantly increased mutation frequencies compared with spermatogenic cells from young or middle-aged mice. Finally, the mutation frequency was observed to increase during spermiogenesis in postreplicative cell types when spermatogenic cells were obtained from old mice.From a genetic perspective, germ cells are profoundly different from somatic cells because they carry the genetic information that will direct the development of the next generation, not simply the next daughter cell. Thus, safeguarding the integrity of germ-line DNA might provide evolutionary advantages. Indeed, in mice, mutation frequencies obtained from mixed populations of germ cells are lower than for somatic tissues (1). This was demonstrated by using a transgenic system in which the bacteriophage genome carrying the lacI repressor gene and the ␣lacZ gene from the prokaryotic lac operon was introduced into the mouse genome as a transgene. DNA was recovered from genomic DNA preparations by packaging and used to infect a strain of Escherichia coli carrying a lacZ (␤-galactosidase) gene, but lacking a functional lacI gene. Mutation of the lacI gene renders a blue plaque on agarose containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-gal). In contrast, other studies using a lacI transgenic mouse (2) or a lacZ transgenic mouse (3) did not report a significant difference in mutation frequencies for spermatogenic cells compared with somatic cells.Although provocative, interpretation of the results demonstrating a lower mutation frequency for male germ cells is complicated by the fact that adult seminiferous tubules contain a mixture of spermatogenic cell types encompassing all stages of spermatogenesis. Spermatogonia serve as the stem cells for spermatogenesis and undergo mitotic divisions that give rise to cells that will either retain their identity as spermatogonia to maintain the stem cell population or enter meiosis to become primary spermatocy...

show abstract

Sources of variability in data from a lacl transgenic mouse mutation assay

Cited by 61 publications

References 17 publications

Detection of extremely rare alleles by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification (Bi-PAP-A): measurement of mutation load in mammalian tissues

Detection of extremely rare alleles by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification (Bi-PAP-A): measurement of mutation load in mammalian tissues

PAP: Detection of ultra rare mutations depends on P* oligonucleotides: ?Sleeping Beauties? awakened by the kiss of pyrophosphorolysis

Mutation frequency declines during spermatogenesis in young mice but increases in old mice

Contact Info

Product

Resources

About