Rapid identification of RT-PCR clones containing translation-terminating mutations

Binnie, Cameron G.; Kam-Morgan, Lauren; Cayouette, Matthew C.; Marra, Giancarlo; Boland, C. Richard; Luce, Michael C.

doi:10.1016/s1383-5718(96)00104-0

Cited by 9 publications

(5 citation statements)

References 15 publications

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“…Analysis of HNF1a expression on the transcriptional level frequently revealed HNF1a transcripts in the tumor tissues (see below). This allowed us to search for HNF1a mutations also at the RNA level by using PTT to locate potential nonsense mutations in HNF1a mRNA [29]. We restricted the analysis to eight tumors and the corresponding normal tissues, as the RNA of only these preparations was of sufficient quality.…”

Section: Resultsmentioning

confidence: 99%

Loss of HNF1α function in human renal cell carcinoma: Frequent mutations in the VHL gene but not the HNF1 gene

Lemm,

Lingott,

Strandmann

et al. 1999

Mol. Carcinog.

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Human renal cell carcinoma (RCC) is a common malignant disease of the kidney characterized by dedifferentiation of renal epithelial cells. Our previous experiments showed that most RCCs have a loss of function of the tissue-specific transcription factor hepatocyte nuclear factor (HNF) 1alpha. Detailed analyses of the 10 exons encoding HNF1alpha in 32 human RCCs by single-strand conformation polymorphism analysis and direct DNA sequencing revealed no tumor-associated mutation, whereas with the same probes we frequently found mutations in the von Hippel-Lindau tumor suppressor gene. No mutation leading to loss of HNF1alpha function was detected by analyzing the integrity of the HNF1alpha transcripts in the RNA derived from RCCs by the protein truncation test. Investigating human RCC cell lines by western blotting and gel retardation assays showed a dramatic loss in the expression of the tissue-specific transcription factor HNF1alpha in eight of 10 cell lines. As the HNF1alpha-related transcription factor HNF1beta was expressed in all these tumor cell lines, the loss of HNF1alpha expression was a specific event and was maintained in RCC cell lines. The loss of HNF1alpha expression in RCC cell lines on the RNA level was confirmed by reverse transcription polymerase chain reaction. We propose that tumor-associated mutations in the HNF1alpha gene do not occur in human RCC and that the loss of function is partially due to a transcriptional inactivation of the HNF1alpha gene.

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Section: Resultsmentioning

confidence: 99%

Loss of HNF1α function in human renal cell carcinoma: Frequent mutations in the VHL gene but not the HNF1 gene

Lemm,

Lingott,

Strandmann

et al. 1999

Mol. Carcinog.

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“…Plasmid-derived IVTT polypeptides were judged to comigrate with either the normal or the truncated protein by gel electrophoresis, and only cDNA prepared from colonies giving rise to truncated protein were sequenced. 34 Direct sequencing of cloned cDNA was performed by automated methods with the use of either fluorescein-labeled dideoxy terminators (Applied Biosystems) or Sequenase, version 2.0 (U.S. Biochemical). Mutations were confirmed in genomic DNA derived from leukemic cells by amplifying the relevant exon with the use of primers described elsewhere 35 and performing cloning and sequencing reactions as described above.…”

Section: Methodsmentioning

confidence: 99%

Homozygous Inactivation of theNF1Gene in Bone Marrow Cells from Children with Neurofibromatosis Type 1 and Malignant Myeloid Disorders

Side¹,

Taylor²,

Cayouette³

et al. 1997

N Engl J Med

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277

171

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“…For example, mutations in the promoter and 3′-untranslated regions of MSH2 and MLH1 could can not be detected. However, sequencing of individual exons of genomic DNA or in vitro transcription/ translation assays have similar limitations [Kohonen-Corish et al, 1996;Biennie et al, 1997;Lin et al, 1999].…”

Section: Discussionmentioning

confidence: 99%

Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes

et al. 2000

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The most sensitive technique for the detection of germline mutations is exon by exon sequencing of the gene under investigation using genomic DNA as a template for analysis. This approach, however, has cost and sensitivity limitations that can, at least in part, be overcome by RNA‐based analysis. Germline mutations of MLH1 and MSH2 are the most frequent cause of the inherited susceptibility to colorectal and other epithelial cancers known as hereditary non‐polyposis colorectal cancer (HNPCC). We compared the analysis of the MLH1 and MSH2 genes using mRNA and genomic DNA as starting material from 21 HNPCC patients. All samples were investigated by RT‐PCR, sequencing of cDNA and simultaneous sequencing of genomic DNA. The cDNA was generated using specific primers complementary to the ends of MLH1 and MSH2 genes, respectively. Mutations in MLH1 and MSH2 were detected in 11 out of 21 unrelated patients. In 10 out of 11 cases, mutations were detected independently of the type of primers used for reverse transcription (RT). One novel missense mutation (K751R) in MLH1 was detected using this method. One nonsense mutation (E205X) in MSH2 was only detectable when RT was performed using MSH2 gene‐specific primers. Shorter PCR products indicative of alternatively spliced transcripts were not observed when MLH1 or MSH2 specific cDNA RT primers were employed to generate template, except in one case where exon skipping was observed for exons 9 and 10. In this report we demonstrate that primers specific for RT of MLH1 and MSH2 are crucial for increasing the sensitivity of cDNA analysis. DNA sequencing using RNA as a basis for template construction may be a valuable and economical alternative to genomic DNA sequencing. Hum Mutat 17:52–60, 2001. © 2001 Wiley‐Liss, Inc.

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Rapid identification of RT-PCR clones containing translation-terminating mutations

Cited by 9 publications

References 15 publications

Loss of HNF1α function in human renal cell carcinoma: Frequent mutations in the VHL gene but not the HNF1 gene

Loss of HNF1α function in human renal cell carcinoma: Frequent mutations in the VHL gene but not the HNF1 gene

Homozygous Inactivation of theNF1Gene in Bone Marrow Cells from Children with Neurofibromatosis Type 1 and Malignant Myeloid Disorders

Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes

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