PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 9436045

Nonradioisotopic telomeric repeat amplification protocol (TRAP) using digoxigenin labeled probe.

Y Wada·A Yagihashi·H Kameshima·A Matsuura·N Sato·K Kikuchi·T Yajima·K Sasaki·N Uehara·N Watanabe·K Hirata
View original source ↗Export metadata ↓

Abstract

We have attempted to detect telomerase activity by Southern blot analysis using a digoxigenin labeled probe (5'-(CCCTTA)6CCCTAA-3') Telomerase activity was detected in all cancer cell lines that were tested, and:the lower limit for detection was 10(3) cells. Although telomerase activity was detected in colon cancer tissues, it was not found in adenoma or normal tissues. Telomerase activity was completely abrogated by heat and RNase treatment of the CHAPS extract. Our results demonstrate that this nonradioisotopic assay is safe and reliable for detecting telomerase activity in cancer cell lines and biopsy samples.

Explore related subjects

Blotting, SouthernDigoxigeninFemaleGene AmplificationHumansPolymerase Chain ReactionRepetitive Sequences, Nucleic AcidSensitivity and SpecificityTelomeraseTumor Cells, CulturedJournal Article

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Y Wada, A Yagihashi, H Kameshima, A Matsuura, N Sato, K Kikuchi, T Yajima, K Sasaki, N Uehara, N Watanabe, K Hirata. 1997. Nonradioisotopic telomeric repeat amplification protocol (TRAP) using digoxigenin labeled probe.. https://doi.org/10.3109/08923979709007667

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2012

Limitations of encapsidation of recombinant self-complementary adeno-associated viral genomes in different serotype capsids and their quantitation.

We previously reported that self-complementary adeno-associated virus (scAAV) type 2 genomes of up to 3.3 kb can be successfully encapsidated into AAV2 serotype capsids. Here we report that such oversized AAV2 genomes fail to undergo packaging in other AAV serotype capsids, such as AAV1, AAV3, AAV6, and AAV8, as determined by Southern blot analyses of the vector genomes, although hybridization signals on quantitative DNA slot-blots could still be obtained. Recently, it has been reported that quantitative real-time PCR assays may result in substantial differences in determining titers of scAAV vectors depending on the distance between the primer sets and the terminal hairpin structure in the scAAV genomes. We also observed that the vector titers determined by the standard DNA slot-blot assays were highly dependent on the specific probe being used, with probes hybridizing to the ends of viral genomes being significantly overrepresented compared with the probes hybridizing close to the middle of the viral genomes. These differences among various probes were not observed using Southern blot assays. This overestimation of titer is a systemic error during scAAV genome quantification, regardless of viral genome sequences and capsid serotypes. Furthermore, different serotypes capsid and modification of capsid sequence may affect the ability of packaging intact, full-length AAV genomes. Although the discrepancy is modest with wild-type serotype capsid and short viral genomes, the measured titer could be as much as fivefold different with capsid mutant vectors and large genomes. Thus, based on our data, we suggest that Southern blot analyses should be performed routinely to more accurately determine the titers of recombinant AAV vectors. At the very least, the use of probes/primers hybridizing close to the mutant inverted terminal repeat in scAAV genomes is recommended to avoid possible overestimation of vector titers.

Blotting, Southern↗
PubMed2007

Random segment deletion based on IS31831 and Cre/loxP excision system in Corynebacterium glutamicum.

A simple and random genome deletion method combining insertion sequence (IS) element IS31831 and the Cre/loxP excision system generated 42 Corynebacterium glutamicum mutants (0.2-186 kb). A total of 393.6 kb (11.9% of C. glutamicum R genome) coding for 331 genes was confirmed to be nonessential under standard laboratory conditions. The deletion strains, generated using only two vectors, varied not only in their lengths but also the location of the deletion along the C. glutamicum R genome. By comparing and analyzing the generated deletion strains, identification of nonessential genes, the roles of genes of hitherto unknown function, and gene-gene interactions can be easily and efficiently determined.

Blotting, Southern↗
PubMed2007

Analysis of plasmids encoding extended-spectrum beta-lactamases (ESBLs) from Escherichia coli isolated from non-hospitalised patients in Seville.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli is an emerging pathogen in the community. We investigated the specific types of ESBLs in E. coli isolates from non-hospitalised patients and the plasmids harbouring these ESBLs. Forty-eight consecutive ESBL-producing E. coli were studied. All the isolates were clonally unrelated. ESBLs were characterised by sequencing. Mobility of the undigested plasmids was investigated both by conventional electrophoresis and by pulsed-field gel electrophoresis. Southern restriction fragment length polymorphism (RFLP) experiments were carried out by digesting plasmids with EcoRI and HindIII. CTX-M-14 was the most frequent ESBL (28 isolates), followed by SHV-12 (16 isolates). Plasmids encoding CTX-M-14 appeared homogeneous both in hybridisation profile and mobility. In contrast, plasmids expressing SHV-12 were more varied in size and hybridisation patterns. Our data suggest that a single type or very closely related plasmids harbouring CTX-M-14 are spreading in the community among unrelated E. coli isolates in our area.

Blotting, Southern↗