Fluorescence-based method for measuring and determining the mechanisms of recombination in quantitative PCR

Shammas, Fuad V.; Heikkilä, Reino; Osland, Arve

doi:10.1016/s0009-8981(00)00374-0

Cited by 29 publications

(25 citation statements)

References 14 publications

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“…Second, to explore the notion that secondary structure in the template DNA was contributing to our results, we repeated both the E. coli rRNA and the seal mRNA using standard PCRs in the presence of DMSO and determined the rf values. DMSO is often used in PCR specifically to render amplifiable difficult templates, such as those with secondary structure (20,21). However, DMSO did not noticeably lower the rf in either system (Table 3).…”

Section: Resultsmentioning

confidence: 99%

Detection of High Levels of Recombination Generated During PCR Amplification of RNA Templates

Rusterholtz

Krummel

et al. 2006

BioTechniques

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Recombination during the PCR amplification of DNA templates can be a serious problem for those seeking to genotype heterogeneous populations, yet a boon to those seeking to enhance variation during in vitro evolution. Here, the extent to which PCR generates chimeric full-length products was estimated using a powerful restriction fragment-length polymorphism (RFLP) assay involving the use of fluorescently labeled PCR primers. Three different RNA-encoding DNA templates were assayed: (i) one for a group I ribozyme, (ii) one for a 16S ribosomal RNA (rRNA), and (iii) one for a messenger RNA (mRNA). In all cases, the observed frequency of chimeric PCR products exceeded 20%, and longer templates appear to produce more chimeric products. Although two of these templates have the potential to form secondary structures during the PCR, this tendency does not seem to heighten recombination frequency. These results corroborate previous studies that show that the production of chimeras can be best attenuated to a certain extent by varying the extension times in PCR.

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Section: Resultsmentioning

confidence: 99%

Detection of High Levels of Recombination Generated During PCR Amplification of RNA Templates

Rusterholtz

Krummel

et al. 2006

BioTechniques

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“…The commercially available AMV and MMlV reverse transcriptase have the ability to switch from one template to another resulting in recombinant DNA artefacts (9,10). In addition, thermostable polymerases generate artificial recombinant fragments by template switching (11)(12)(13)(14). Thus, any amplified PCR product deviated from the anticipated ones should be considered with great caution.…”

Section: Discussionmentioning

confidence: 99%

Absence of the JAZF1/SUZ12 chimeric transcript in the immortalized non-neoplastic endometrial stromal cell line T HESCs

Panagopoulos

2010

Oncology Letters

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Abstract. Endometrial stromal sarcomas are rare malignancies, accounting for less than 10% of uterine sarcomas. The most characteristic chromosomal aberration of this tumor type is the translocation t(7;17)(p15-p21;q12-q21) leading to the fusion of two zinc finger genes, JAZF1 and SUZ12. Recently, the presence of the neoplastic JAZF1/SUZ12 fusion transcript was reported in normal cells of human endometrium. One of the positive samples for the JAZF1/SUZ12 transcript was the immortalized T HESCs cell line. This cell line was derived from the stromal cells obtained from an adult female with myomas and immortalized by transfection of a human telomerase gene. Since T HESCs has a normal karyotype and no fusion of the two genes occurs at the genomic level, the JAZF1/SUZ12 transcript was proposed to be generated by regulated trans-splicing between precursor RNAs for JAZF1 and SUZ12. However, no confirmatory reports currently exist. To determine whether the results could be reproduced, the T HESCs cell line was subjected to three different RT-PCR amplifications for the JAZF1/SUZ12 fusion transcript. RT-PCR assays did not amplify JUZF1/SUZ12 cDNA fragments in the T HESCs cell line, whereas the same assays easily generated JUZF1/SUZ12-amplified transcripts in an endometrial stromal cell sarcoma carrying the t(7;17) chromosomal aberration. Thus, the presence, if any, of a JUZF1/SUZ12 chimeric transcript in the immortalized normal T HESCs is not a constant, reproducible result. IntroductionEndometrial stromal sarcomas (ESS) are rare malignancies, accounting for less than 10% of uterine sarcomas (1). The most characteristic chromosomal aberration of this tumor type is the translocation t(7;17)(p15-p21;q12-q21) leading to the fusion of two zinc finger genes, JAZF1 (juxtaposed with another zinc finger) and SUZ12 (also known as JJAZ1) (2). Recently, Li et al (3) reported the presence of the neoplastic JAZF1/SUZ12 fusion transcript in normal cells of human endometrium. One of the samples found to be positive for the JAZF1/SUZ12 transcript was the immortalized T HESCs cell line. This cell line was derived from the stromal cells obtained from an adult female with myomas and immortalized by transfection of a human telomerase gene (4). Since this cell line has a normal karyotype and no fusion of the two genes occurs at the genomic level, it was proposed that the JAZF1/ SUZ12 transcript in the T HESCs cells is generated from regulated trans-splicing between precursor RNAs for JAZF1 and SUZ12 (3). However, no confirmatory reports exist thus far. To determine whether the results obtained by Li et al (3) could be reproduced, the T HESCs cell line was subjected to various RT-PCR for the JAZF1/SUZ12 fusion transcript. Materials and methods Materials. The cell line T HESCs was an American TypeCulture Collection (ATCC) culture (ATCC-CRL-4003; Lot No. 3857441) purchased from LGC Promochem (http://www. lgcpromochem-atcc.com/) and cultured according to ATCC recommendations. Thus, the cells were grown in DMEM/ F12 medium with 3.1 g/l glucose, 1 ...

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“…targeted mutagenesis or targeted gene repair of cells. [Liu et al, 1997a;Barnard et al, 1998] Number of thermal cycles and amount of templates in PCR and/or + + Correction factor (k) SNuPE reaction [Liu et al, 1997b;Shammas et al, 2001] Non-linearity of peak heights on a LIF-CE instrument +/-+ Calibration curve Biased PCR and/or SNuPE amplification towards one allele due to -+ Calibration curve unequal amount of allele-specific templates [Walsh et al, 1992] …”

Section: Discussionmentioning

confidence: 99%

Quantification of single nucleotide polymorphisms: A novel method that combines primer extension assay and capillary electrophoresis

et al. 2001

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We present a novel method for accurate quantification of single nucleotide polymorphism (SNP) variants in transcripts and pooled DNAs in a one-tube reaction. Our approach is based on single- nucleotide primer extension (SNuPE) and laser-induced fluorescence capillary electrophoresis (LIF-CE), and takes advantage of distinct mobilities of SNuPE products with different nucleotides incorporated at their 3' ends. The method, called SNuPE-ONCE, was tested on two polymorphisms and five mutations that comprised the three most frequent ( approximately 70%) nucleotide changes in the human genome (C/T, A/G, and A/T). The usefulness of the method was demonstrated by analyzing nonsense-mediated mRNA instability in fibroblasts. Our data show 1) that the method provides highly reproducible relative allele frequencies (SD<0.017) with a good accuracy (e.g. for heterozygotes 0.500 +/- 0.036, P = 0.01), depending on the sequence and the proportion of the SNP variants in the sample, and 2) that relative allele frequencies as low as 1% can be detected quantitatively and unambiguously. Our assay relies on a CE instrument available in many laboratories and offers a useful method for quantitative SNP genotyping as well as for a variety of expression studies.

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Fluorescence-based method for measuring and determining the mechanisms of recombination in quantitative PCR

Cited by 29 publications

References 14 publications

Detection of High Levels of Recombination Generated During PCR Amplification of RNA Templates

Detection of High Levels of Recombination Generated During PCR Amplification of RNA Templates

Absence of the JAZF1/SUZ12 chimeric transcript in the immortalized non-neoplastic endometrial stromal cell line T HESCs

Quantification of single nucleotide polymorphisms: A novel method that combines primer extension assay and capillary electrophoresis

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