Genotypic analysis of multiple loci in somatic cells by whole genome amplification

Barrett, Michael T.; Reid, Brian J.; Joslyn, Geoffrey

doi:10.1093/nar/23.17.3488

Cited by 34 publications

(24 citation statements)

References 14 publications

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“…In our experience this marks the lowest limit of cell numbers for homogeneous biallelic amplification and lies slightly below the value reported by Barrett et al (50-to 100-cell equivalents). 19 However, in contrast to our studies, Barrett et al used DNA dilution series. Thus, cell lysis has not been considered in their studies.…”

Section: Discussionmentioning

confidence: 59%

“…These techniques have recently been extended for the use in tumor pathology where preneoplastic and neo-plastic fluorescence-activated cell sorter (FACS)-sorted aneuploid esophageal tumor cells were analyzed for microsatellite instability. 5,19 In contrast to single specific PCR 20 and subchromosomal analysis by in situ hybridization 21 this procedure allows multiple molecular analyses in only few or even single tumor cells.…”

mentioning

confidence: 99%

“…The number of cells is especially critical for both microsatellite instability (MSI) and LOH detection because too few cells or too little DNA may result in unequal allelic amplification. 13,19,23,31 Barret et al 5 examined 1000 flow-sorted esophageal tumor cells rather than single cells to prevent an artifactual dysequilibrium between the two different allelic fragments due to low cell number. The limiting cell number of microdissected tissue sections has not been systematically investigated for MSI or LOH analyses.…”

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confidence: 99%

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Multiple Mutation Analyses in Single Tumor Cells with Improved Whole Genome Amplification

Dietmaier

Hartmann

Wallinger

et al. 1999

The American Journal of Pathology

213

159

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Section: Discussionmentioning

confidence: 59%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Multiple Mutation Analyses in Single Tumor Cells with Improved Whole Genome Amplification

Dietmaier

Hartmann

Wallinger

et al. 1999

The American Journal of Pathology

213

159

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“…To screen the p16 locus (9p21) for possible homozygous deletions or allelic loss, DNA from clonal cell populations was amplified by primer extension preamplification (PEP) (5,60). Aliquots from each sample were evaluated for STS marker c5.1, located within the p16 gene (29), or polymorphic markers D9S942 and D9S161 in locus-specific PCRs as described previously (6).…”

Section: Methodsmentioning

confidence: 99%

Inactivation of p16 in Human Mammary Epithelial Cells by CpG Island Methylation

Foster

Wong

Barrett

et al. 1998

Molecular and Cellular Biology

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211

178

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Proliferation of human mammary epithelial cells (HMEC) is limited to a few passages in culture due to an arrest in G 1 termed selection or mortality stage 0, M0. A small number of cells spontaneously escape M0, continue to proliferate in culture, and then enter a second mortality stage, M1, at which they senesce. Evidence that M0 involves the Rb pathway comes from the observation that expression of human papillomavirus type 16 E7 alleviates the M0 proliferation block, and we further show that the Rb-binding region of E7 is required to allow cells to bypass M0. In contrast, E6 does not prevent HMEC from entering M0 but, rather, is involved in M1 bypass. Here we show that inactivation of the D-type cyclin-dependent kinase inhibitor p16INK4A is associated with escape from the M0 proliferation block. Early-passage HMEC express readily detectable amounts of p16 protein, whereas normal or E6-expressing HMEC that escaped M0 expressed markedly reduced amounts of p16 mRNA and protein. This initial reduction of p16 expression was associated with limited methylation of the p16 promoter region CpG island. At later passages, a further reduction in p16 expression occurred, accompanied by increased CpG island methylation. In contrast, reduction of p16 expression did not occur in E7-expressing HMEC that bypassed M0, due to inactivation of Rb. These observations in the E6-expressing HMEC correlate well with the finding that CpG island methylation is a mechanism of p16 inactivation in the development of human tumors, including breast cancer.

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“…DNA was extracted from the flow-purified cell populations using either standard phenol-chloroform or the Puregene DNA Isolation kit as recommended by the manufacturer (Gentra Systems, Inc., Minneapolis, MN). Whole genome amplification using primer extension preamplification was performed as described previously (57,61) for each sorted fraction and three constitutive controls per participant.…”

Section: Patients (Research Participants)mentioning

confidence: 99%

Selectively Advantageous Mutations and Hitchhikers in Neoplasms

et al. 2004

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Neoplastic progression is an evolutionary process characterized by genomic instability and waves of clonal expansions carrying genetic and epigenetic lesions to fixation (100% of the cell population). However, an evolutionarily neutral lesion may also reach fixation if it spreads as a hitchhiker on a selective sweep. We sought to distinguish advantageous lesions from hitchhikers in the premalignant condition Barrett's esophagus. Patients (211) had biopsies taken at 2-cm intervals in their Barrett's segments. Purified epithelial cells were assayed for loss of heterozygosity and microsatellite shifts on chromosomes 9 and 17, sequence mutations in CDKN2A/MTS1/INK4a (p16) and TP53 (p53), and methylation of the p16 promoter. We measured the expanse of a lesion in a Barrett's segment as the proportion of proliferating cells that carried a lesion in that locus. We then selected the lesion having expanses >90% in the greatest number of patients as our first putative advantageous lesion. We filtered out hitchhikers by removing all expanses of other lesions that did not occur independent of the advantageous lesion. The entire process was repeated on the remaining expanses to identify additional advantageous lesions. p16 loss of heterozygosity, promoter methylation, and sequence mutations have strong, independent, advantageous effects on Barrett's cells early in progression. Second lesions in p16 and p53 are associated with later selective sweeps. Virtually all of the other lesion expansions, including microsatellite shifts, could be explained as hitchhikers on p16 lesion clonal expansions. These techniques can be applied to any neoplasm.

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Genotypic analysis of multiple loci in somatic cells by whole genome amplification

Cited by 34 publications

References 14 publications

Multiple Mutation Analyses in Single Tumor Cells with Improved Whole Genome Amplification

Multiple Mutation Analyses in Single Tumor Cells with Improved Whole Genome Amplification

Inactivation of p16 in Human Mammary Epithelial Cells by CpG Island Methylation

Selectively Advantageous Mutations and Hitchhikers in Neoplasms

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