The discrimination of high-risk HPV types by in situ hybridization and the polymerase chain reaction

Summary The parameter Tm^t has been defined by non-isotopic in situ hybridization and describes the tissue melting temperature (Tm^t) of human papillomavirus (HPV) DNA sequences. In this study, multiple in situ hybridization signals for HPV types 16, 31 and 33 in individual archival biopsies hybridized with genomic probes are shown by polymerase chain reactions to be due to cross-hybridization of probe sequences to a single tissue target. Tm^t is independent of viral type but depends on the homology between probe and target when using nick-translated whole genomic probes. The difference between Tm and Tm^t is not due to the presence of viral capsid protein. Multiple HPV signals in archival material should not therefore be interpreted as indicative of multiple HPV infection unless adequate stringency conditions have been employed or they are present in morphologically distinct areas of the biopsy.Furthermore, extrapolation of calculated DNA homologies to non-isotopic in situ hybridization analysis may not be appropriate. A hybridization signal does not imply probe and target identity: this has implications for HPV typing in clinical material.     

Detection of human papillomavirus in cervical scrapings by in situ hybridization and the polymerase chain reaction in relation to cytology

Summary A gynaecological out-patient population consisting of 200 patients aged 19–43 years (mean age 34.2 years) was screened for the presence of human papillomavirus (HPV) by the polymerase chain reaction and in situ hybridization on cervical scrapings. A novel method was applied for the detection of HPV in cervical cells by embedding them in a paraffin block before in situ hybridization was performed. This technique resulted in well preserved cytological morphology, easy performance and economy of probes. In eight of the 200 patients (4%), human papillomavirus DNA was revealed by the polymerase chain reaction. Subtyping revealed the presence of HPV serotype 16 DNA in three of these patients. In one patient HPV serotype 18 DNA was also present. The in situ hybridization assay was able to detect all those cases with a specific HPV serotype infection.     

原位PCR技术及其应用前景

原位PCR是分子生物学领域中一种崭新的技术,它结合了PCR技术和原位杂交技术的优点.该文介绍了原位PCR技术的起源、发展及方法学,并简要描述了该技术的应用现状及前景．     

Re-usable DNA template for the polymerase chain reaction.

DNA covalently bound to an uncharged nylon membrane was used for consecutive amplifications of several different genes by PCR. Successful PCR amplifications were obtained for membrane-bound genomic and plasmid DNA. Membrane-bound genomic DNA templates were re-used at least 15 times for PCR with specific amplification of the desired gene each time. PCR amplifications of specific sequences of p53, p16, CYP1A1, CYP2D6, GSTM1 and GSTM3 were performed independently on the same strips of uncharged nylon membrane containing genomic DNA. PCR products were subjected to restriction fragment length polymorphism analysis, single-strand conformational polymorphism analysis and/or dideoxy sequencing to confirm PCR-amplified gene sequences. We found that PCR fragments obtained by amplification from bound genomic DNA as template were identical in sequence to those of PCR products obtained from free genomic DNA in solution. PCR was performed using as little as 5 ng genomic or 4 fg plasmid DNA bound to membrane. These results suggest that DNA covalently bound to membrane can be re-used for sample-specific PCR amplifications, providing a potentially unlimited source of DNA for PCR.     

Fluorescein as a label for non-radioactive in situ hybridization

Summary Non-radioactive techniques can be applied to many in situ hybridization (ISH) applications, and a number of non-radioactive labels for this process have been reported. However, these labels have some inherent problems in terms of both background and signal-to-noise values. We have sought to address these issues by searching for an alternative label that has the following features: efficient incorporation into probes, non-endogenous to biological systems, the availability of a high-affinity, high-specificity antibody. Fluorescein has been shown to meet these requirements. In addition, due to the fluorescent nature of the label, it has been possible to design a rapid, non-radioactive labelling assay and also to view in situ hybridization results by direct fluorescence in certain ISH applications. The hybridization kinetics have been investigated. Significant improvements have been made to the hybridization buffer leading to reduced background and increased rates of hybridization when compared to traditional hybridization buffers.     

Urine as another potential source for template DNA in polymerase chain reaction (PCR)

This technical note examines the potential for preparing template DNA in polymerase chain reactions (PCR) from urine in Japanese macaques (Macaca fuscata). Microsatellite band patterns from urine samples showed close agreement with those of blood and fecal samples, and only a few hundred μl of urine yielded a template DNA for PCR. This research will increase the opportunity for scientists to examine the genetic backgrounds of their target animals by using non‐invasive sample collection in the wild. Am. J. Primatol. 48:299–304, 1999. © 1999 Wiley‐Liss, Inc.     

Polymerase chain reaction in situ: an appraisal of an emerging technique

Summary Polymerase chain reaction (PCR)in situ is a new technique which promises to enhance considerably our ability to detect a few copies of target nucleic acid sequences in fixed tissues and cells. It has an enormous potential for application in diagnostic histopathology of viral diseases and in the study of gene expression. PCRin situ is, however, technically difficult, and amplification of the target DNA is only 30–300 fold. In this article we present an overview of PCRin situ techniques used to amplify both DNA and RNA targets (RT-PCRin situ). We also identify problems which can reduce the efficiency of the technique or which can give rise to false-positive results. They include (1) the inhibitory effects of cross-linking of histones to DNA or PCR amplification, (2) abstraction of PCR reagents by tissue-bonding agents which are used to coat glass slides, (3) poor denaturation of target DNA and subsequent DNA renaturation due to extensive cross-linking of histones to DNA, or because of incorrect temperature regulation of thermal cyclers, (4) false-positive results which arise from end-labelling of DNA strand breaks byTaq polymerase, and (5) diffusion of PCR products into and out of cells leading to false-positive results. We present some of the approaches that have been used to overcome some of these difficulties and suggest new avenues for investigation to improve this technique further.     

基于荧光原位杂交的藜属植物核型分析

为精确地识别藜属植物染色体组的核型特征,该文研究了4种来自青海高原的野生藜属植物(灰绿藜、藜、菊叶香藜及杂配藜)和1种从美国引进的栽培藜麦品种PI614932-HX(3)基于染色体荧光原位杂交(rDNA FISH)的核型。利用5S rDNA和45S rDNA对5种藜属植物有丝分裂中期的染色体进行FISH研究。藜属植物的核型分析结果表明:(1)藜属植物中存在二倍体(2n=2x=18)和四倍体(2n=4x=36)两种倍性,藜麦和灰绿藜为四倍体,其余3种为二倍体。(2)藜麦、灰绿藜、藜、菊叶香藜及杂配藜的核型公式分别为2n=4x=36=34m(2AST)+2sm,2n=4x=36=32m(4AST)+4sm,2n=2x=18=16m(4AST)+2sm,2n=2x=18=18m及2n=2x=18=16m+2sm。(3)染色体由大部分的中部着丝粒染色体(m)和少部分近中部着丝粒染色体(sm)组成。(4)核型类型除了菊叶香藜为1B以外,其余均属于2B类型。(5)在藜麦、灰绿藜及藜中具有分布位置不同、数量不等的双随体。5S rDNA、45S rDNA FISH结果表明:(1)藜麦和灰绿藜的染色体上存在2对5S rDNA位点和1对45S rDNA位点,藜、杂配藜的染色体上存在1对5S rDNA位点和1对45S rDNA位点,菊叶香藜的染色体上只存在1对5S rDNA位点。(2)5S rDNA和45S rDNA位点均位于染色体的短臂上。该研究首次获得了藜属植物基于5S rDNA和45S rDNA荧光原位杂交核型,为藜属植物亲缘关系研究和细胞生物学研究提供了分子细胞遗传学依据。     

Karyotyping of Brassica oleracea L. based on rDNA and Cot -1 DNA fluorescence in situ hybridization

To explore an effective and reliable karyotyping method in Brassica crop plants, Cot-1 DNA was isolated from Brassica oleracea genome, labeled as probe with Biotin-Nick Translation Mix kit, in situ hybridized to mitotic spreads, and where specific fluorescent bands showed on each chromosome pair. 25S and 5S rDNA were labeled as probes with DIG-Nick Translation Mix kit and Biotin-Nick Translation Mix kit, respectively, in situ hybridized to mitotic preparations, where 25S rDNA could be detected on two chromosome pairs and 5S rDNA on only one. Cot-1 DNA contains rDNA and chromosome sites identity between Cot-1 DNA and 25S rDNA was determined by dual-colour fluorescence in situ hybridization. All these showed that the karyotyping technique based on a combination of rDNA and Cot-1 DNA chromosome landmarks is superior to all but one. A more exact karyotype of B. oleracea has been analyzed based on a combination of rDNA sites, Cot-1 DNA fluorescent bands, chromosome lengths and arm ratios. __________ Translated from Journal of Wuhan University (Nat. Sci. Ed.), 2006, 52(2): 230–234 [译自: 武汉大学学报 (理学版)]     

Extraction of DNA from exfoliative cytology specimens and its suitability for analysis by the polymerase chain reaction

The extraction of DNA from archival exfoliative cytology samples would allow the molecular biological analysis of this readily available material using the polymerase chain reaction (PCR). We have quantitatively and qualitatively studied the extraction of DNA from a variety of cytological preparations. For both fresh and archival cervical smears, overnight incubation with proteinase K produces high yields of high molecular weight DNA, but simply boiling the samples produces DNA suitable for PCR amplification of a single copy gene. Increasing the proteinase K incubation to several days allows the extraction of DNA from fixed and stained archival cytology slides from a variety of sites. The extracted DNA was again suitable for PCR analysis. Fresh and archival cytological material can be utilized for molecular biological study of disease processes using PCR. Archival cytological material is probably the best source of DNA and RNA after stored frozen tissue.     

Spatial analysis of intranuclear human repetitive DNA regions by in situ hybridization and digital fluorescence microscopy

Summary Non-isotopic (fluorescent) in situ hybridization has established itself as a useful technique for the localization of DNA sequences in both metaphase and interphase cells. The rapid development of digital fluorescence microscopy, especially confocal microscopy, has become a powerful aid for the evaluation of the hybridization results in cytogenetic and cell biological applications. In this review we will demonstrate the utility of these methodologies for the three-dimensional visualization and analysis of chromosome-specific (peri)centromeric repetitive DNA sequences within the intranuclear structure of human cells and cell lines.     

Enzymatic detection systems for non-isotopic in situ hybridization using biotinylated cDNA probes

Summary The feasibility of various non-isotopic enzymatic detection systems was tested for in situ hybridization using biotin-labelled, nick-translated cDNA probes. For this purpose, we isolated and prepared cDNA restriction fragments encoding the proteolytic cysteine proteinase cathepsin L and analysed Kirsten murine sarcoma virus-transformed BALB/3T3 cells, which have been shown to express high amounts of cytoplasmic RNA of this ras oncogene-induced proteinase. When compared on a semiquantitative basis, colorimetric non-isotopic detection of cDNA hybrids with avidin-biotin-peroxidase conjugates visualized by silver intensification of the nickel-diaminobenzidine end-product was superior to that obtained with avidin-biotin-alkaline phosphatase using different substrates for development. When the peroxidase staining technique was applied for RNA detection, it was found that overnight incubation in methanol containing hydrogen peroxide followed by deproteination with HCl was the most effective method for inhibition of endogenous peroxidase activity. For DNA detection, non-specific nucleic staining was completely abolished when heat treatment (100°C) of the cell specimens was performed prior to hybrpidization.     

Localization of mRNA for testis-specific histone H1t by in situ hybridization

H1t is a testis-specific H1 histone variant that appears in germ cells during the meiotic prophase of mammalian spermatogenesis. Using a tritiated antisense RNA probe, H1t mRNA was identified by in situ hybridization in the mid and late pachytene spermatocytes found in seminiferous tubules of approximately stages VII to XIII.