单纯疱疹病毒2型特异性DNA片段的克隆和鉴定

用核酸限制性内切酶BamHI对单纯疱疹病毒2型(HSV—2)的DNA进行酶解,回收位于基因组中的反向重复序列区的Bam HIG片段,然后将其克隆在载体质粒PUC 8的Bam HI切点上,进一步用核酸限制性内切酶Eco RI和KPNI对这一重组质粒联合酶解,移去EcoRI—KPNI小片段,经末端修饰后,将其连接得到新的重组质粒pRC102,它含有一小段HSV—2的DNA序列。以此质粒为探针,分别与HSV—1、HSV—2及细胞DNA进行斑点杂交;与HSV—1和HSV—2酶解后的DNA片段进行Southern转印系交。两组实验结果显示,pRC102质粒DNA只与HSV—2 DNA特异性杂交,其HSV—2的型特异性良好。     

菠菜叶绿体DNA 4.1 kb的SalⅠ片段的克隆

利用pBR 322衍生质粒DNA为载体,将菠菜叶绿体DNA的SalⅠ限制性片段插入质粒的Sal Ⅰ位点,获得52个含重组质粒的菌落(Ap~rTc~5)。并对每个克隆的质粒进行限制性内切酶分析,通过Southern吸印与探针杂交,证明了重组质粒pSS104含有的插入DNA是菠菜叶绿体DNA 4.1kb的SalⅠ片段。迄今在该片段尚未定位任何已知的叶绿体基因,用大肠杆菌的活体系统也未能发现这段DNA的转录产物,本文对此进行了讨论。     

一种简单有效的克隆未知旁侧DNA片段的方法

操作含长插入片段的DNA克隆时 ,经常需要进行亚克隆和测序实验。通常的方法首先是得到插入片段的限制性内切酶谱 ,然后选择合适的内切酶消化DNA ,分离靶片段 ,将其连接入质粒载体中进行下一步操作。但这种方法工作量大 ,步骤繁琐。在此 ,介绍一种不需要做限制性内切酶谱分析 ,而根据靶片段的旁侧序列直接进行亚克隆实验的方法。首先 ,选择合适的限制性内切酶消化含长插入片段的DNA克隆 ,其中一种酶切在已知的旁侧序列上 ,另一为随机选择 ;然后酶切混合物与线性化的质粒载体连接 ,转化细菌得到一“亚克隆库” ;将其中的克隆挑选入 96孔板培养后 ,按行或列混合菌液得到相应的“pool” ;最后 ,用PCR方法筛选获得含靶DNA片段的阳性克隆 ,其中所用的引物一个与已知的旁侧DNA序列配对 ,另一个与质粒载体上序列配对 ,PCR扩增已知的旁侧DNA片段以鉴定阳性克隆。多次独立实验表明该方法简单有效 ,可广泛用于亚克隆和DNA步移实验     

金霉素链霉菌启动基因在大肠杆菌中的克隆及表达

利用质粒pGA46作为载体,将金霉素链霉菌染色体DNA的Sau3A酶切片段插入pGA-46的BglII位点,获得氯霉素抗性、四环素抗性的重组质粒。DNA同源性分析表明,重组质粒中外源序列来自金霉素链霉菌DNA。重组质粒的限制性酶切产物电泳分析,可以重新找到载体DNA片段。将链霉菌的插入序列移入另一载体pBR322中,仍可表现启动基因的功能。用限制性内切酶酶切及核酸酶BAL-31酶切等方法,已将链霉菌的插入序列降至200bp以下,仍保留启动基因功能。     

平末端DNA随机连接构建重组质粒

目的:拼接DNA片段并克隆。方法:用T4DNA连接酶将DNA片段以平末端随机连接,随后用限制性内切酶切割,琼脂糖电泳分离酶切产物,挑选特定片段纯化回收,与线性化的载体质粒连接,转化大肠杆菌感受态细胞。结果:通过以上步骤,成功拼接了不同DNA片段,构建了含有目的拼接片段的重组质粒。结论:该方法简便、易行、可靠,可作为拼接、克隆DNA的备选方案,在分子生物学研究和基因工程中应用。     

油桐尺蠖核型多角体病毒DNA片段的分子克隆及其鉴定

使用限制性核酸内切酶 BamHI 将油桐尺蠖核型多角体病毒(Buzura SuppressariaNuclear Polyhedrosis Virus。简称 BsNPV)DNA 切为9个片段。通过聚丙烯酰胺凝胶电泳洗脱,将其酶切片段与 BamHI 切割后的质粒 pBR_(322)重组,转化大肠杆菌,经抗菌素筛选、电泳分折和菌落杂交3种方法证明了插入子的存在,从而表明油桐尺蠖核型多角体病毒 DNA BamHI 片段已经克隆。     

亚洲小车蝗Oedaleus asiaticus痘病毒DNA探针的构建和初步应用(英文)

用限制性内切酶酶切亚洲小车蝗痘病毒DNA,将酶切后的DNA片断克隆到质粒pGEM—7zf( )中。用随机引物法标记克隆的外源亚洲小车蝗痘病毒DNA片断为探针。经Southern及斑点杂交证明此探针对新疆戟纹蝗Dociostaurus kraussi及微孢子虫Nosema locusstae有特异性,对新疆意大利蝗Calliptomus italicus无特异性。构建的探针可检测1ng亚洲小车蝗痘病毒DNA及10~2病毒包含体,也可检测感染病虫匀浆液中的痘病毒。     

肠杆菌膜蛋白基因的研究——Ⅳ.大肠杆菌染色体DNA Hind Ⅲ酶解片段上

大肠杆菌野生株JE5506(1pp⁺)和突变株JE5505(1pp^-)的染色体DNA的Hind Ⅲ酶解片段,与一带有大肠杆菌外膜脂蛋白信号肽基因的107bp探针,在20℃下进行DNA-DNA杂交,在25kb和3.4kb处各出现一杂交带。该两片段与载体质粒pBR322在体外进行DNA重组,分别得到pHWO14和pHWO15两个重组质粒。该两重组质粒的限制性内切酶酶切图谱,Southern印迹及与107bp探针杂交的实验结果,进一步证明了上述两个DNA片段上存在有与     

谷氨酸棒状杆菌启动子在枯草芽孢杆菌中的克隆及其序列分析

利用枯草芽孢杆菌启动子探针质粒pPL 603为载体,从谷氨酸棒状杆菌1014—6染色体DNA上克隆到一个有较强启动功能的DNA片段。生物素标记的DNA—DNA分子杂交实验证明该片段确实来自谷氨酸棒状杆菌1014—6染色体DNA。在绘制该片段限制酶酶切图谱的基础上,经另一个启动子探针质粒pPL 703的亚克隆,将其启动子功能区定位在Bamm酶切片段中。对后者进行了核苷酸序列分析,发现它具有棒状杆菌类启动子的－35区和－10医序列。     

龙葵叶绿体ATP合酶α-亚单位基因的克隆

本研究以菠菜叶绿体DNA 2.45kb的SalI片段(含有ATP合酶α-亚单位基因)为探针,从龙英叶绿体DNA BamHI片段文库中筛选出含龙葵叶绿体atpA基因的克隆。通过Southrcn吸印与探针杂交,证明了重组质粒pSB 132的插入片段含有atpA基因。同时将atpA基因定位在龙葵叶绿体DNA SalI、BglI、XhoI和BamHI 4种酶切图谱的限制性片段上。     

Presence of human papillomavirus genome in human tumor cell lines and cultured cells.

A series of 47 human carcinoma cell lines and their cultured cells were examined for human papillomavirus (HPV) genomes with the use of an HPV detection kit (DNA-RNA hybridization, mixed HPV DNA probe of types 6, 11, 16, 18, 31, 33 and 35). Four of 8 cases of mild dysplasia, 3 of 9 cases of severe dysplasia, 3 of 7 cases of carcinoma in situ, 3 of 15 cases of uterine carcinoma and 5 of 6 cases of condyloma acuminatum were shown to contain the HPV DNA genome in primary cultured cells, while HPV was not detected in the third-passage cells except for the three cases of large cell, nonkeratinizing squamous cell carcinoma. HPV was also not detected in such normal tissues as uterine cervical squamous epithelium, uterine cervical columnar epithelium and endometrium. The presence of HPV DNA genomes was detected consistently in the passages of three lines (SKG-II, HKMUS and HKTUS; large cell nonkeratinizing squamous cell carcinomas of the uterine cervix) with the use of the Southern Blot method (DNA-DNA hybridization, mixed HPV probe of types 6, 11, 16 and 18). HPV type 16 DNA was detected in HKTUS, and HPV type 18 DNA was found in SKG-II and HKMUS. The other 44 cell lines, including ovarian carcinoma, endometrial carcinoma, sarcoma, gastric cancer, pancreatic cancer and rectal cancer, were negative for the HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33 and HPV-35 genomes under stringent hybridization conditions.     

Integration of human papillomavirus type 16 DNA sequences: a possible early event in the progression of genital tumors.

The keratinocyte line SK-v harbors only integrated human papillomavirus type 16 (HPV 16) DNA sequences, although it originated from vulvar Bowenoid papules predominantly containing multiple copies of free HPV 16 genomes. We have cloned a fragment of cell DNA that contains the integrated HPV 16 DNA sequences and have shown that integration interrupts the HPV 16 genome in open reading frames E2 and L2 and creates a deletion of 813 base pairs. This allows the expression of open reading frames E6 and E7, as actually substantiated by Northern (RNA) blot analysis of SK-v RNAs with subgenomic HPV 16 RNA probes. Using a unique flanking cellular DNA sequence as the probe, we have shown that the integration of HPV 16 sequences had already occurred in the premalignant lesions from which the SK-v cell line was derived.     

Detection and quantification of hepatopancreatic parvovirus in penaeid shrimp by real-time PCR assay

As one of the major pathogens, hepatopancreatic parvovirus (HPV) can cause severe diseases in penaeid shrimp. We developed a TaqMan-based real-time PCR assay for the HPV detection in China. A pair of primers (HPVF and HPVR) and a TaqMan probe were designed according to the HPV genomic sequence of Chinese isolate (GenBank: GU371276). Our data showed that the primers and TaqMan probe were specific for HPV, and they exhibited no cross-reaction with infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV) and specific pathogen free (SPF) shrimp DNA. The assay had a detection limit of four plasmid HPV DNA copies per reaction. Furthermore, HPV was detected in 16 of 21 Fenneropenaeus Chinensis, 3 of 52 Litopenaeus vannamei and 2 of 2 Marsupenaeus japonicus penaeid shrimp samples. In addition, HPV was also detected in crabs. Therefore, this assay could be successfully used as a sensitive and rapid molecular-based diagnostic method to screen HPV-free animals and survey the prevalence of HPV in cultured populations of penaeid shrimp in China.     

Study of stringency conditions for human papillomavirus DNA detection on cell lines,frozen and paraffin-embedded tissue sections by in situ hybridization with biotinylated probes

Summary In situ hybridization was mainly used for typing human papillomavirus (HPV) in paraffin-embedded or frozen sections under stringent conditions (SC). We tested 5 different conditions of stringency with biotinylated HPV 1, 2, 16 and 18 probes on 3 cell lines (Siha and CaSki with HPV16, HeLa with HPV18) by varying the concentration of formamide in the hybridization mixture and washings in order to determine the stringency conditions to be used to assess the presence of HPV and its typing: A-low stringency, hybridization at 35° C below the melting temperature of DNA (Tm-35° C) and washings without formamide; B-low stringency, hybridization and washings at Tm-35° C; C-medium stringency, hybridization at Tm-35° C and washings at Tm-12° C; D-high stringency, hybridization at Tm-12° C and washing without formamide; E-very high stringency, hybridization and washings at –12° C. This study showed that HPV typing required a high stringency. On the contrary, under non stringent conditions (NSC), each cell line was positive with the heterologous probes.When 3 to 5 stringency conditions were assayed on 4 frozen samples, similar results were obtained. Typing required high stringency conditions whereas NSC allowed HPV detection. Furthermore, this study demonstrated the specificity of the reaction in lesions positive with more than one type.Stringent (Tm-12° C) and non stringent (Tm-35° C) conditions of hybridization were further applied to 57 biopsy sections (17 frozen and 40 paraffin-embedded specimens) from typical wart lesions and lesions suspected of HPV. Nineteen samples were totally negative under both NSC and SC, and considered as non-infected by HPV. In 22 specimens positive, under both NSC and stringent conditions (SC), the HPV type was identified. Ten specimens reacted with 1, 2 or 3 HPV types under NSC but the HPV DNA was not typed with the probes used. Six lesions were negative under NSC but were typed under SC. Most paraffin sections were labeled only with one HPV probe under NSC, whereas frozen sections were often labeled with 2 or 3 HPV probes. The HPV probe positive under SC was usually positive under NSC in both frozen and paraffin sections. HPV type 1 probe was more frequently positive under NSC in paraffin- embedded sections than the others and the 4 probes tested were equally positive in frozen sections.These findings show the interest of in situ hybridization in low stringency conditions since 17% of our lesions (10/57) were positive only under NSC: HPV DNA was detected but not typed with the probes used. Frozen sections were more frequently positive than paraffin sections, suggesting a loss of DNA accessibility in the latter, due to the fixation or processing before hybridization.     

Detection of hepatopancreatic parvovirus in Thai shrimp Penaeus monodon by in situ hybridization,dot blot hybridization and PCR amplification

Hepatopancreatic parvovirus (HPV) infects the hepatopancreas in penaeid shrimp and retards their growth. The DNA sequence of HPV from Thai shrimp Penaeus monodon (HPVmon) differs from HPV of Penaeus chinensis (HPVchin) by approximately 30%. In spite of this difference, commercial PCR primers (DiagXotics) developed from HPVchin to yield a 350 bp PCR product do give a 732 bp product with HPVmon DNA template. On the other hand, the sensitivity of HPVmon detection with these primers and with hybridization probes designed for HPVchin is significantly lower than it is with HPVchin. To improve sensitivity for HPVmon detection, we used the sequence of the 732 bp HPVmon PCR amplicon described above to develop specific PCR primers (H441F and H441R) and hybridization probe. The primers could detect as little as 1 fg of purified HPVmon DNA while the 441 bp digoxygenin-labeled PCR product gave strong, specific reactions with in situ hybridization and with hybridization blots. In contrast, negative results were obtained using DNA from all other pathogens tested and from DNA of P. monodon. Supernatant solution from boiled, fresh shrimp fecal and postlarval samples homogenized in 0.025% NaOH/0.0125% SDS could be used to detect as little as 0.1 pg HPVmon DNA by the PCR reaction. By dot blot hybridization, a visible signal was obtained with purified HPVmon DNA at 0.01 pg, but detection in spiked feces and postlarval samples was only 1 and 0.1 pg, respectively.     

Detection of HPV DNA in cervical specimens collected in cytologic solution by ligation-dependent PCR

OBJECTIVE: To determine the feasibility and sensitivity of detecting human papillomavirus (HPV) in specimens collected in Cytyc PreservCyt fluid (Boxborough, Massachusetts, U.S.A.) using ligation-dependent polymerase chain reaction (LD-PCR) and to demonstrate the diagnostic value of HPV DNA testing as an adjunct to cytology in the detection of cervical squamous intraepithelial lesions (SIL), especially in cases of atypical squamous cells of undetermined significance (ASCUS). STUDY DESIGN: LD-PCR is a recently invented DNA amplification technology that utilizes a capture probe for target isolation and 2 hemiprobes for target detection. The hemiprobes are designed in such a way that when they hybridize to their target, the 5' end of one probe and the 3' end of the other probe are brought together. Two hemiprobes can then be ligated into a full probe that can serve as a template for PCR amplification. A total of 94 cervical specimens were collected in cytologic fluid and tested with LD-PCR. The results were compared with those of the Digene Hybrid Capture II assay (HC II) (Beltville, Maryland, U.S.A.) and consensus PCR. RESULTS: The overall sensitivity for detecting HPV was 41.5% (39/94) by LD-PCR, 50% (47/94) by consensus PCR and 37.2% (35/94) by HC II. The prevalence of HPV by HC II, consensus PCR and LD-PCR were 87.5%, 100% and 87.5% in the high grade SIL group; 100%, 90.9% and 90.9% in the low grade SIL group; 30%, 52.5% and 40% in the ASCUS group; and 14.2%, 22.8% and 17.1% in women with normal cytology. These results indicate that all 3 methods have similar sensitivity in patients with SIL. However, there is greater variation in detection rates in the ASCUS and normal cytology groups. CONCLUSION: LD-PCR is a useful method of detecting HPV in liquid-based gynecologic cytologic preservatives, and HPV testing as a method adjunct to the liquid-based Pap test could be useful in detecting SILs, especially for the management of patients with ASCUS.     

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.     

Rapid detection of human papilloma virus using a novel leaky surface acoustic wave peptide nucleic acid biosensor

A novel leaky surface acoustic wave (LSAW) bis-peptide nucleic acid (bis-PNA) biosensor with double two-port resonators has been constructed successfully for the quantitative detection of human papilloma virus (HPV). The bis-PNA probe can directly detect HPV genomic DNA without polymerase chain reaction (PCR) amplification, and it can bind to the target DNA sequences more effectively and specifically than a DNA probe. When the concentrations varied from 1 pg/L to 1000 μg/L, with 100 μg/L being the optimal, a typical linearity was found between the quantity of target and the phase shifts. The detection limit was 1.21 pg/L and the clinical specificity was 97.22% of that of real-time PCR. The bis-PNA probe was able to distinguish sequences that differ only in one base. Both the intraassay and interassay coefficients of variance (CVs) were <10%, and the biosensor can be regenerated for ten times without appreciable loss of activity. Therefore, this technical platform of LSAW biosensor can be applied to clinical samples for direct HPV detection.     

Detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp by in situ hybridization at the electron microscope level

A post-embedding in situ hybridization procedure was developed to detect hepatopancreatic parvovirus (HPV) of penaeid shrimp at the ultrastructural level. The procedure was optimized using sections of resin-embedded hepatopancreas from HPV-infected juvenile Penaeus monodon and postlarval P. chinensis. The hepatopancreata were fixed using various fixatives, dehydrated, and embedded in the hydrophilic resin Unicryl. A 592 bp HPV-specific DNA probe, labeled with DIG-11-dUTP, was tested both on semi-thin and ultra-thin sections and examined by light and electron microscopy, respectively. Hybridized probe was detected by means of an anti-DIG antibody conjugated to 10 nm gold particles and subsequent silver enhancement. Hybridization signal intensities were similar with all fixatives tested, but ultrastructure was best preserved with either 2 or 6% glutaraldehyde. Post-fixation with 1% osmium tetroxide improved ultrastructure but markedly decreased hybridization signal and induced non-specific deposition of gold and silver. Under optimized conditions, this technique was used to successfully follow the development of HPV from absorption and transport through the cytoplansm to nuclear penetration, replication and release by cytolysis. The probe signal was consistently observed among necrotic cell debris within the lumen of hepatopancreatic tubules, within the microvillous border of tubule epithelial cells, within the cytoplasm, and within diagnostic HPV intranuclear inclusion bodies. The nucleolus and karyoplasm of patently infected cells (i.e., showing HPV intranuclear inclusion bodies) were almost devoid of signal. Electron-lucent structures, known as intranuclear bodies, commonly found within the virogenic stroma, showed only weak labeling. This is the first use of in situ hybridization to detect HPV nucleic acids with the electron microscope. The technique should be useful for studying the pathogenesis of HPV.     

Human Papillomavirus Detection In Cervical Cells By In Situ Hybridization With Biotinylated Probes

We have investigated the applicability of human papillomavirus (HPV) DNA detection by in situ hybridization with biotinylated probes in epithelial cells obtained from the cervix using a cotton tip swab. We describe a simple procedure for obtaining homogeneous cell samples and good preservation of cellular structure. This is achieved by pretreatment of cells with L-cysteine before hybridization. Separate denaturation of cellular DNA and probe DNA is also necessary for satisfactory results. Both benign HPV DNA 6/11 and potentially oncogenic HPV DNA 16/18 could be identified in our series. In situ hybridization on cervical scrapes is a rapid, simple and very specific method for detecting patients infected with oncogenic HPV types.