ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Cytogenetics came of age of 1970, when the first banding technique was applied to human chromosomes, and the technique has proved invaluable in diagnosis of malignancy since that time. However. while karyotype analysis is extremely useful, it is labor-intensive and requires years of specialist training. By the mid 1980s the field was ripe for labor and time-saving methods. The natural route was to combine cytogenetics with speedy molecular techniques. This led to the development of fluorescent in situ hybridization (FISH). In FISH a piece of DNA, tagged in such a way that it can be detected at a later time, is used as a probe. The probe and the target [a chromosome preparation containing metaphase spreads and interphase (nondividing) nuclei on a microscope slide] are denatured so that the DNA of each is single-stranded. The probe is added to the chromosome preparation (in situ). The slide is incubated or hybridized for long enough for the probe to match up with the target, usually overnight (hybridization). Any unbound probe is removed by washing after the incubation period. Washing conditions are finely tuned to remove any probe that bound to other sequences due to weak sequence similarity The location of the probe is highlighted through the use of a fluorescent dye (fluorescent). The combination of cytogenetic and molecular techniques has proved extremely powerful and has led to the resolution of many questions that could not have been answered by other methods.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Recent evidence suggests that the tumor suppressor protein, p53, protects somatic cells against the accumulation of genomic mutations. The genomes of cells lacking normal p53 function may become hypermutable, a condition that might result in the accumulation of multiple genetic alterations as the affected cells proliferate. Such cells may then become more susceptible to malignant transformation. We hypothesized that some high-grade prostate cancers might arise from foci of morphologically benign cells that had previously sustained p53 lesions. As an initial test of this hypothesis, we employed a microdissection technique to isolate morphologically benign cells within hyperplastic glands located near foci of high-grade adenocarcinoma. Genomic DNA from these cells was subjected to polymerase chain reaction amplification and single-stranded conformational polymorphism analysis for detecting alterations in the p53 locus. With use of this approach, gross alterations in the p53 locus were demonstrated in benign cells in 1 of 20 (5%) specimens harboring high-grade malignancy (Gleason grade 7 or higher). Thus, in some cases, hyperplastic prostatic epithelium harbors preneoplastic genetic alterations that could possibly give rise to high-grade malignancies.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Archival pathological specimens are a source of RNA and DNA for clinical surveillance or retrospective studies. We employed a modification of the acid guanidium thio-cyanate-phenol-chloroform extraction method for the recovery of total RNA from formalin-fixed, paraffin-embedded neoplastic thyroid tissue. The extracted RNA was used for reverse transcription ofptcand subsequent amplification of the complementary DNA (cDNA) by the reverse transcription-polymerase chain reaction (RT-PCR). In lieu of32P-labeled DNA for hybridization studies, we supplemented the nucleotide pool in the amplification reaction with a modified pyrimidine. digoxigenin-11-dUTP Digoxigenin-11-dUTP was incorporated directly into the PCR product, eliminating the need for hybridization, posthybridization washes, and prolonged autoradiography. These products were resolved by electrophoresis on agarose gels. Southern blotted to nylon membranes, and rapidly detected by chemiluminescence. This nonradioisotopic method has expedited and reduced the cost for molecular investigations with archival pathological specimens by providing equal sensitivity to or greater sensitivity than that of DNA-labeled radionuclides without the associated biological hazards

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The prognostic significance of p53 immunoreactivity in adult patients with supratentorial fibrillary astrocytic neoplasms was examined by Kaplan-Meier survival analysis. Using a monoclonal antibody that reacts with both mutant and wild-type p53 protein (PAb 1801), reactivity was assessed immunohistochemically in specimens from the first diagnosis of astrocytic neoplasm in 95 patients: 26 astrocytomas (A), 19 anaplastic astrocytomas (AA). and 50 glioblastomas multiforme (GBM). Overall, 53% of cases exhibited any p53 nuclear immunoreactivity, with approximately the same proportion in each histologic grade. Survival was measured from diagnosis to death or last follow-up and ranged from 3 months to 9 years. Histologic grade was a powerful prognostic variable for this group of patients (p < 0.001), with median survivals of 88, 18, and 9 months for A, AA, and GBM patients, respectively. In contrast, patients with p53-immunoreactive or -nonimmunoreactive neoplasms had median survival times of 18 or 15 months, respectively (p = 0.21). These results indicate that p53 immunoreactivity was not prognostically significant in this group of adult patients with supratentorial fibrillary astrocytic neoplasms, although a small difference in survival cannot be excluded.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

In order to evaluate the feasibility of using quantitative polymerase chain reaction (PCR) to evaluate gene dosage, we developed an assay to detect α-globin genes, which are frequently deleted in α-thalassemia patients. In this quantitative assay α-1 and α-2 globin consensus regions are coamplified by one oligonucleotide pair, along with a second primer pair targeting a single-copy reference gene, namely, tumor necrosis factor alpha, or TNF-α. A series of DNA samples titrating α-globin against TNF-α DNA have a strong linear relationship between template ratios and product ratios (r > 0.98). Minimal sequence divergence (919f homology) between α-1 and α-2, internal to the identical primer annealing sites, results in a lower amplification efficiency for α-1, to 94% of α-2 for each cycle. Furthermore, when applied to a variety of individual DNA samples, the signal ratios of α-globin to TNF-α were far more variable than previously observed for titrated control DNA. We conclude that DNA isolates from different individuals may have idiosyncratic changes in amplification efficiency owing to polymorphic sequence variation and/or variable presence of unidentified contaminants. Despite these potentially confounding factors, however, we were able to identify by quantitative PCR a single gene deletion later confirmed by Southern blot analysis in 20 individual DNA samples.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The cyclin Dl gene, located on chromosome llql3, is frequently rea%anged in parathyroid neoplasms and amplified in some carcinomas of other organs. Recent studies have detected amplification of cyclin Dl and other markers on chromosome 1 lql3 (evaluated by Southern or slot blot assays) in ∼25–50% of squamous cell carcinomas of the esophagus and noted that amplification was associated with lessened survival time. We applied the technique of differentia] polymerase chain reaction to the evaluation of cyclin Dl gene amplification in squamous cell carcinomas of the esophagus. Cyclin Dl was found to be amplified in 10 of 45 (22%) primary tumors and three of 12 (25%) lymph node metastases. Lymph node metastases tended to be more common in patients with cyclin Dl amplification (70%) than in those without amplification (37%). In 36 patients with follow-up. cyclin Dl amplification was associated with decreased 1 year survival (28% vs. 59%). Cyclin Dl gene amplification in esophageal carcinomas can be evaluated by differential polymerase chain reaction and may provide useful prognostic information.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Triplex-polymerase chain reaction technique (PCR) was developed for the detection and identification of mycobacterial DNA sequences in uncultured clinical samples. A 123 bp fragment corresponding to a specificMycobacterium tuberculosissequence complex, a 383 bp DNA fragment encoding for part of the 65 kD mycobacterial surface antigen, and a 268 bp fragment of the human (3-globin gene to demonstrate the presence of suitable DNA were amplified by triplex PCR. To demonstrate the applicability of this method, 206 alcohol-fixed, paraffin-embedded sputum samples from 47 patients with culture-proven tuberculosis were investigated. Of 206 samples, 157 were PCR positive, resulting in correct diagnosis of tuberculosis in 46 of 47 (97.8%) patients. Furthermore, 165 alcohol-fixed, auramin-stained sputum smears were examined in a blind trial. Triplex PCR revealed tuberculosis in 20 of 21 samples from patients with tuberculosis. In comparison, cultures were positive in 20 of 21 samples. and acid-fast organisms were found by microscopy in 18 of 21 samples. We conclude that triplex PCR is a rapid and sensitive technique for the detection of mycobacterial DNA in uncultured clinical samples and offers equivalent sensitivity (95.2%) and specificity (98.6%) as do culture methods.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

We have examined the loss of heterozygosity (LOH) of codon 72 and evaluated the overexpression of the tumor suppressor genep53in 43 primary human prostatic adenocarcinomas (PC). DNA from tumors and normal tissues were extracted from radical prostatectomy specimens. LOH was determined by restriction fragment length polymorphism analysis (RFLP) of the codon-specific endonuclease-digested polymerase chain reaction (PCR) products. Results showed 17 heterozygous cases (39%) among this patient group. Seven of the heterozygous cases displayed LOH. Six of the seven LOH cases were high-grade PCs with Gleason's combined score of ≥7 and showed capsular invasion. One of the LOH cases, however, displayed an intermediate morphological score of 6 but also with evidence of capsular invasion. The 43 primary PCs were also examined for over-expression ofp53by a monoclonal antibody-mediated immunofluorescence reaction. Overexpression of nuclearp53as detected by antibody was demonstrable only in tumors with combined morphological Grade ≥7. No significant overexpression ofp53was noted in lower-grade tumors. In addition. 10 cases of benign prostatic hyperplasia (BPH) were evaluated forp53expression. All 10 cases showed no detectablep53overexpression.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Molecular analyses to determine clonality of T and B cells in malignant lymphoma and leukemia and to detect the (9;22) translocation in chronic myelogenous leukemia are commonly used in clinical molecular biology laboratories. We describe the inclusion of a sensitivity control in each of these assays derived from DNA of well-characterized cell lines. The inclusion of such a sample adds an important quality-control parameter to ensure assay-to-assay reproducibility and to satisfy accreditation and regulatory requirements.

ISSN:1052-9551    

出版商:OVID    

年代:1994

数据来源: OVID