Loss of Heterozygosity Assay for Molecular Detection of Cancer Using Energy-transfer Primers and Capillary Array Electrophoresis

Medintz, Igor L.; Lee, Chyi‐Chia Richard; Wong, Wendy W.; Pirkola, Kristin; Sidransky, David; Mathies, Richard A.

doi:10.1101/gr.10.8.1211

Cited by 50 publications

(31 citation statements)

References 12 publications

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“…This demonstrates the feasibility of collecting large amounts of data on a very rapid time scale using radial CAE chip analysis. Some of these samples had been analyzed previously on the Molecular Dynamics 96-capillary MegaBACE 1000 instrument (Medintz et al 2000b); the MegaBACE analysis took 75 min as opposed to the 9.5 min demonstrated here. Each sample analyzed on the CAE microplates consisted of a complex mixture of ladder and multiple differentially labeled amplicons, and yet single-base-pair resolution was achieved.…”

Section: Discussionmentioning

confidence: 99%

High-Performance Multiplex SNP Analysis of Three Hemochromatosis-Related Mutations With Capillary Array Electrophoresis Microplates

Medintz¹,

Wong²,

Berti³

et al. 2001

Genome Res.

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An assay is described for high-throughput single nucleotide polymorphism (SNP) genotyping on a microfabricated capillary array electrophoresis (CAE) microchip. The assay targets the three common variants at the HFE locus associated with the genetic disease hereditary hemochromatosis (HHC). The assay employs allele-specific PCR (ASPCR) for the C282Y (845g->a), H63D (187c->g), and S65C (193a->t) variants using fluorescently-labeled energy-transfer (ET) allele-specific primers. Using a 96-channel radial CAE microplate, the labeled ASPCR products generated from 96 samples in a reference Caucasian population are simultaneously separated with single-base-pair resolution and genotyped in under 10 min. Detection is accomplished with a laser-excited rotary four-color fluorescence scanner. The allele-specific amplicons are differentiated on the basis of both their size and the color of the label emission. This study is the first demonstration of the combined use of ASPCR with ET primers and microfabricated radial CAE microplates to perform multiplex SNP analyses in a clinically relevant population

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Section: Discussionmentioning

confidence: 99%

High-Performance Multiplex SNP Analysis of Three Hemochromatosis-Related Mutations With Capillary Array Electrophoresis Microplates

Medintz¹,

Wong²,

Berti³

et al. 2001

Genome Res.

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“…Genotypes were determined using Genetic Profiler version 2.0 software and AI detected using the formula (T1/T2)/(N1/N2) where T1 and N1 represent the peak heights of the less intense alleles and T2 and N2 represent the peak heights of the more intense alleles of the tumor and referent samples, respectively (23). Each chromosomal region was represented by 2 polymorphic markers and AI was defined according to the following criteria: (i) when at least one marker for a given region showed an allelic ratio of 0.35 or less, the region was considered to show AI, (ii) when neither marker had an allelic ratio of 0.35 or less and at least one marker was informative, the region was considered normal, and (iii) when both markers were homozygous, the region was considered uninformative.…”

Section: Ai Analysismentioning

confidence: 99%

Genomic (In)stability of the Breast Tumor Microenvironment

Rummel

Valente

Kane

et al. 2012

Molecular Cancer Research

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The breast tumor microenvironment plays an active role in tumorigenesis. Molecular alterations have been identified in tumor-associated stroma; however, there is considerable debate as to whether the stroma is characterized by genomic instability or whether detection of chromosomal alterations reflects technological artifact rather than the true genomic content of the tumor microenvironment. Thus, breast stroma specimens from 112 women undergoing reductive mammoplasty (n ¼ 7), prophylactic mastectomy (n ¼ 6), or mastectomy for a breast disease (n ¼ 99) were frozen in optimal cutting temperature medium. Allelic imbalance (AI) analysis was conducted using a panel of 52 microsatellite markers in 484 stromal specimens from 98 women, of which 92% had no detectable AI events. When compared with previously generated AI data from 77 formalin-fixed, paraffinembedded (FFPE) stroma specimens, 42% of which harbored at least one detectable AI event, the frequency of AI in the FFPE specimens (4.62%) was significantly higher (P < 0.001) than that found in frozen specimens (0.45%). This comparison of AI between FFPE and research-grade specimens suggests that past reports of AI in breast stroma reflect artifact in the archival specimens caused by formalin-fixation, paraffin-embedding and tissue storage. Furthermore, SNP data were generated from a subset of 86 stromal specimens using SNP arrays and copy number alterations were identified using Partek Genomics Suite. For 95% of the specimens, no detectable copy number alterations were found and the 11 changes that were detected were small and not shared between specimens. These data, therefore, support a model in which the tumor microenvironment is genetically stable.

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“…4). AI was detected using the formula (T1 / T2) / (N1 / N2) where T1 and N1 represent the peak heights of the less intense alleles and T2 and N2 represent the peak heights of the more intense alleles of the tumor and referent samples, respectively (29).…”

Section: Methodsmentioning

confidence: 99%

Allelic Imbalance in Primary Breast Carcinomas and Metastatic Tumors of the Axillary Lymph Nodes

Ellsworth

Ellsworth²,

Neatrour³

et al. 2005

Molecular Cancer Research

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Axillary lymph node status is the most important prognostic factor in predicting disease outcome in women with breast cancer. A number of chromosomal aberrations in primary breast tumors have been correlated with lymph node status and clinical outcome, but chromosomal changes particular to metastatic lymph node tumors have not been well studied. DNA samples isolated from laser-microdissected primary breast and metastatic axillary lymph node tumors from 25 women with invasive breast cancer were amplified using 52 microsatellite markers defining 26 chromosomal regions commonly deleted in breast cancer. Levels and patterns of allelic imbalance (AI) within and between breast and lymph node tumors were assessed to identify chromosomal alterations unique to primary or metastatic tumors and to examine the timing of metastatic potential. The overall frequency of AI in primary breast tumors (0.24) was significantly greater (P < 0.001) than that in lymph node tumors (0.10), and congruent AI events were observed for <20% of informative markers. AI at chromosomes 11q23.3 and 17p13.3 occurred significantly more frequently (P < 0.05) in primary breast tumors alone; no chromosomal regions showed a significantly higher AI frequency in lymph nodes. Higher rates of AI in primary versus metastatic lymph node tumors suggest that acquisition of metastatic potential may be an early event in carcinogenesis, occurring before significant levels of AI accumulate in the primary tumor. In addition, patterns of AI were highly discordant between tumor types, suggesting that additional genetic alterations accumulated independently in the two cell populations.

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Loss of Heterozygosity Assay for Molecular Detection of Cancer Using Energy-transfer Primers and Capillary Array Electrophoresis

Cited by 50 publications

References 12 publications

High-Performance Multiplex SNP Analysis of Three Hemochromatosis-Related Mutations With Capillary Array Electrophoresis Microplates

High-Performance Multiplex SNP Analysis of Three Hemochromatosis-Related Mutations With Capillary Array Electrophoresis Microplates

Genomic (In)stability of the Breast Tumor Microenvironment

Allelic Imbalance in Primary Breast Carcinomas and Metastatic Tumors of the Axillary Lymph Nodes

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