Allele dropout in sequential PCR and FISH analysis of single cells (cell recycling)

Rechitsky, Svetlana; Freidine, Michael; Verlinsky, Yury; Strom, Charles M.

doi:10.1007/bf02072532

Cited by 56 publications

(32 citation statements)

References 21 publications

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“…Homozygote PB1s do not exclude the possibility of ADO, are more prone for misdiagnosis, and may require blastomere confirmation. While ADO rates have been reported to be lower in PB analysis than blastomere analysis for many disorders [22][23][24], we did not observe any significant difference in ADO rates in our study. The ADO rates we observed in the actual PGD cases were higher than what we observed in individual fibroblasts (<10%, data not shown).…”

Section: Discussioncontrasting

confidence: 90%

Preimplantation genetic diagnosis (PGD) for nonsyndromic deafness by polar body and blastomere biopsy

Altarescu

Eldar‐Geva

Brooks

et al. 2009

J Assist Reprod Genet

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Purpose Development of an efficient and reliable PGD protocol for nonsyndromic deafness, by polar body (PB) and blastomere PGD. Methods The GJB2/GJB6 mutations along with 12 polymorphic markers were used in PGD analysis of blastomeres or polar bodies in 14 couples for 35 cycles. Marker informativity, diagnosis rates, Allele Drop Out (ADO) rates and PB1 heterozygosity rates were assessed. Results Six cycles were performed by PB biopsy, 27 by blastomere and two combined cycles, resulting in delivery of three unaffected children and five ongoing pregnancies. Diagnosis rates for PB and blastomeres were similar. Only 17% PB1s were heterozygote. ADO rates of 19% were observed in both groups. Conclusions We have developed a single cell multiplex PGD protocol for nonsyndromic deafness with a high efficiency of diagnosis. Most PB1 are homozygous, and similar ADO rates were observed; therefore, blastomere biopsy appears to be the method of choice for this autosomal recessive disease.

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Section: Discussioncontrasting

confidence: 90%

Preimplantation genetic diagnosis (PGD) for nonsyndromic deafness by polar body and blastomere biopsy

Altarescu

Eldar‐Geva

Brooks

et al. 2009

J Assist Reprod Genet

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“…Despite its potential, clinical application of cell recycling is not recommended at present since ADO rates are significantly higher with fixed template DNA than those observed using routine single cell protocols. 133 …”

Section: Cell Recyclingmentioning

confidence: 99%

Molecular Diagnostics in Preimplantation Genetic Diagnosis

Thornhill

Snow

2002

The Journal of Molecular Diagnostics

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“…ADO rates between 0% and 83% have been reported and are dependent on the source and quality of the template DNA amplified (Findlay et al, 1995(Findlay et al, , 1998Garvin et al, 1998;Hahn et al, 1998;Rechitsky et al, 1996;Snabes et al, 1994;Wells et al, 1999). As a result of ADO, mutation analysis may fail to detect heterozygous mutations in a significant number of cases.…”

Section: Heinmöller Et Almentioning

confidence: 99%

Toward Efficient Analysis of Mutations in Single Cells from Ethanol-Fixed, Paraffin-Embedded, and Immunohistochemically Stained Tissues

Heinmöller

Liu

Sun

et al. 2002

Laboratory Investigation

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SUMMARY:Only a few studies have demonstrated successful molecular analysis after whole genome amplification using single cells dissected from paraffin-embedded tissues. The results in these studies were limited by low-amplification efficiency and high rates of allele dropout. In the present study, the amplification rate using a thoroughly modified primer extension and preamplification-PCR protocol was improved significantly for single cells microdissected from paraffin-embedded and immunohistochemically stained tissues. Tissue fixation with ethanol (85%) and the addition of 0.2 mmol/L EDTA helped to achieve an amplification rate between 67% (segments 200 to 400 bp) and 72% (segments Ͻ200 bp). Normal tissue sections were immunohistochemically double stained for overabundance of p53 protein and proliferating cell nuclear antigen. Microdissection of single cells was performed with a manual micromanipulator equipped with a Tungsten needle. Sequence analysis of the TP53 gene was performed after improved primer extension preamplification-PCR and multiplex PCR from single microdissected cells. The rate of allele dropout was at least 68%. These technical advances facilitate routine mutation analysis using a single cell or a few cells microdissected from routinely processed paraffin-embedded normal and tumor tissues. Allele dropout still represents a serious problem in single-cell mutation analysis, especially in samples with limited template DNA and prone to DNA damage. (Lab Invest 2002, 82:443-453).

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Allele dropout in sequential PCR and FISH analysis of single cells (cell recycling)

Abstract: Sequential PCR and FISH analysis of single cells remains an attractive possibility. However, until the problem of the increased rate of ADO is resolved, cell recycling should be applied to clinical preimplantation genetic analysis.

Cited by 56 publications

References 21 publications

Preimplantation genetic diagnosis (PGD) for nonsyndromic deafness by polar body and blastomere biopsy

Preimplantation genetic diagnosis (PGD) for nonsyndromic deafness by polar body and blastomere biopsy

Molecular Diagnostics in Preimplantation Genetic Diagnosis

Toward Efficient Analysis of Mutations in Single Cells from Ethanol-Fixed, Paraffin-Embedded, and Immunohistochemically Stained Tissues

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