Paraffin wax-embedded material as a source of DNA for the detection of somatic genetic changes

Loss of genetic material, corresponding to chromosomal deletions, has been detected in a wide range of tumours and may indicate the position of a tumour suppressor gene. In order to identify the position of such a gene more precisely, many tumour samples must be studied until a minimum consensus deletion is characterized. This process is particularly necessary for lung tumours in which the deletion in chromosome 3, seen with such high frequencies in all histological subtypes, is almost always large. We have recently described the use of the polymerase chain reaction (PCR) for restriction fragment length polymorphism (RFLP) analysis of DNA isolated from small bronchial biopsies of lung tumours. In this study we adapted this technique to allow genotyping of DNA isolated from paraffin wax-embedded material (PWEM) microdissected from glass slides. We have investigated 12 lung tumours at polymorphic loci on chromosome 3 and showed allelic loss in all samples. In adapting PCR–RFLP analysis for DNA isolated from PWEM, we have concentrated on those approaches which might be adaptable to routine clinical practice. Somatic genetic changes are now being identified in many tumour types, and this information is expected to be of diagnostic and prognostic significance.     

Genotypic analysis of sequential genital herpes simplex virus type 1 (HSV-1) isolates of patients with recurrent HSV-1 associated genital herpes

Clinical recurrences of Herpes simplex virus type 1 (HSV-1)-associated genital herpes are thought to be caused by reactivation of latent endogenous HSV-1. However, the possibility of reinfection with exogenous HSV-1 cannot be excluded. This study aimed to determine the incidence of genital HSV-1 superinfection in patients by investigating the genotype of sequential HSV-1 isolates obtained from the same anatomical site of patients with clinical recurrences of genital HSV-1 recurrent genital herpes. Sequential genital HSV-1 isolates were genotyped by PCR amplification of the hypervariable regions located within the HSV-1 genes US1 and US12. Whereas the sequential HSV-1 isolates in 11 of the 13 patients studied had the same genotypes, the sequential isolates of 2 patients showed a different genotype. The data suggest that HSV-1-induced recurrent genital herpes can be associated with genital reinfection with an exogenous HSV-1 strain.     

中药“神经再生素”促神经生长过程中基因差异性表达的初步研究

为了探索中药“神经再生素”促神经生长过程中基因水平的变化。本实验采用 dd-PCR方法 ,从体外培养背根神经节细胞加药组和不加药组中获得两者的差异表达片段 ,并经反杂交筛选、克隆测序、DNA序列检索分析、Northern验证。结果表明 ,差异显示共获得 8个差异条带 ,一个为下调基因 ,其余为上调基因 ,其中 2个 c DNA序列与 RRAJ5 16 1基因 (增殖相关基因 )、AF196 3 15基因 (锌指样蛋白 DDP2 ) 10 0 %同源 ,2个 c DNA序列与 AK0 0 175 7基因、STA5 SRR基因 (t RNA合成酶 )部分同源。结论是中药“神经再生素”在促神经生长过程中 ,对神经元基因的选择性表达起着重要的调控作用。     

Setting out the frame conditions for feasible use of FFPE derived RNA

Introduction

The usage of formalin-fixed paraffin embedded (FFPE) tissue is characterized by its long shelf-life and simple handling. Therefore it is the most commonly available tissue specimen in routine diagnostics and histological studies. Formaldehyde fixation may result in RNA degradation and cross linking with proteins, while storage conditions also affect RNA integrity. The present study was designed to investigate the influence of these factors on RNA analysis.

应用IRS-PCR对金黄色葡萄球菌分型的研究

目的探讨低频限制性位点聚合酶链反应(infrequent-restriction-site PCR，IRS-PCR)在金黄色葡萄球菌(简称金葡菌)基因分型中的应用价值。方法建立本实验室IRS-PCR方法。同时用IRS-PCR和脉冲场凝胶电泳(PFGE)对金葡菌进行基因分型。根据49株社区感染分离菌的分型结果计算辨别力指数(ID)值估计分辨力。对其中30株社区感染菌重复实验一次估计重复性。比较两种基因分型方法的分型率、分辨力、重复性、结果的一致率及操作特点。结果建立IRS-PCR对金葡菌基因分型的方法。70株金葡菌均可被2种方法分型，分型率100％。IRS-PCR分为38个型，21株院内感染菌分为6个型，49株社区感染菌分为32个型，计算ID值为0．981。PFGE分为40个型，21株院内感染菌分为6个型，49株社区感染菌分为34个型，计算ID值为0．983。两种分型方法的重复性均为100％。对院内感染菌，两种方法分型的一致率为100％；对社区感染菌，两种方法分型的一致率为92％(45／49)。与PFGE相比，IRS-PCR更简单、省时、易于操作、不需特殊昂贵仪器。结论IRS-PCR能对金葡菌简易快速可靠分型，适合检验科对临床标本的快速有效分型，是一种有价值的分子流行病学研究工具。     

Real-time PCR检测风疹病毒方法学建立及临床应用

为了建立一种能广泛应用于临床快速、简便、灵敏度和特异性较好的风疹病毒(RV)定量诊断方法。针对RV基因保守序列设计两对引物和一条荧光双标记探针。将PCR扩增所得到的产物片段克隆,作为定量检测的标准品,进行Real-time PCR检测,绘制标准曲线。将此方法和ELISA试剂盒平行检测50份孕妇血清,评估两种方法检出阳性率的差异显著性。Real-time PCR法能较好地检出RVcDNA载量。曲线的相关系数(r)为0.998,可检测线性范围大约在103~109copies/μl,灵敏度接近103copies/μl;批间、批内CV值分别为3.36%、0.94%;也有较好的特异性。与经典的ELISA方法相比,有显著性差异。Real-time PCR方法操作简单、快速,并能避免PCR后处理导致的假阳性污染,实现实时定量。此方法对RV感染的临床诊断和疗效判断等方面有较大的指导意义。     

荧光定量PCR法在重组逆转录病毒pLXSN-BDNF滴度测定中的应用

目的制备含大鼠脑源性神经营养因子（BDNF）基因的逆转录病毒，建立荧光定量PCR测定滴度的方法。方法扩增BDNF基因，构建重组质粒pLXSN-BDNF，将其转染包装细胞，经G418筛选、病毒浓缩后用荧光定量PCR法和克隆形成法测定滴度。结果经PCR、酶切和测序鉴定，BDNF基因成功插入逆转录病毒载体中，筛选得到稳定的分泌重组病毒细胞系。重组病毒100倍浓缩后经荧光定量PCR法和克隆形成法测定的滴度分别为6．92×10^6拷贝/ml和3．2×10^5CFU/ml，两种方法测得的结果差异有统计学意义（P〈0．05）。结论成功扩增BDNF基因，制备了重组病毒pLX—SN-BDNF，建立了荧光定量PCR检测滴度的方法，为基因治疗青光眼性视神经损伤的实验研究提供重要参考指标。     

DNA fingerprinting of sister blastomeres from human IVF embryos

BACKGROUND: Previously published single cell DNA fingerprinting systems have been plagued by high rates of allele drop-out (ADO) and preferential amplification (PA) preventing clinical application in preimplantation genetic diagnosis. METHODS: Tetranucleotide microsatellite markers with high heterozygosity, known allelic size ranges and minimal PCR stutter artefacts were selected for chromosomes X, 13, 18 and 21 and optimized in a multiplex fluorescent (FL)-PCR format. FL-PCR products were analysed using the ABI Prism 377 DNA sequenator and Genescan software. Validation of the DNA fingerprinting system was performed on single diploid (n = 50) and aneuploid (n = 25) buccal cells and embryonic blastomeres (n = 21). RESULTS: The optimized pentaplex PCR DNA fingerprinting system displayed a high proportion of successful amplifications (>91%) and low ADO and PA (<6%) when assessed on 50 human buccal cells. DNA fingerprints of single cells from a subject with Down's syndrome detected the expected tri-allelic pattern for the chromosome 21 marker, confirming trisomy 21. In a blind study on 21 single blastomeres, all embryos were identifiable by their unique DNA fingerprints and shared parental alleles. CONCLUSIONS: A highly specific multiplex FL-PCR based on the amplification of five highly polymorphic microsatellite markers was developed for single cells. This finding paves the way for the development of a more complex PCR DNA fingerprinting system to assess aneuploidy and single gene mutations in IVF embryos from couples at genetic risk.