Objective:To investigate the polyadenylation of mRNA in E. Coli. Methods: The mRNA of E. Coli was enriched from the total RNA with oligo(dT)-cellulose, prior to reverse transcription using oligo(dT)18as the primer. Do...Objective:To investigate the polyadenylation of mRNA in E. Coli. Methods: The mRNA of E. Coli was enriched from the total RNA with oligo(dT)-cellulose, prior to reverse transcription using oligo(dT)18as the primer. Double-stranded cDNA was subsequently synthesized, which was subjected to digestion with Sau3A I to produce multiple gene fragments for ligation with the adapters. PCR was carried out in 10 groups according to 10 different pairs of the selective primers, and the PCR products were then cloned into T-vectors. Results: More than 100 gene fragments had been cloned, 30 of which were sequenced. Conclusion:Polyadenylation of E. Coli mRNA may not be a biochemical curiosity but a general attribute of bacterial mRNA.展开更多
AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was ap...AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.展开更多
Objective To understand the deletion in the survival motor neuron gene (SMN) of childhood onset spinal muscular atrophy (SMA) in Chinese, and the value of diagnosis of SMA using polymerase chain reaction restric...Objective To understand the deletion in the survival motor neuron gene (SMN) of childhood onset spinal muscular atrophy (SMA) in Chinese, and the value of diagnosis of SMA using polymerase chain reaction restriction fragment length polymorphism (PCR RFLP)method. Methods\ Deletions of SMN gene of exon 7 and 8 in 10 cases of presumed SMA, and 20 normal controls from 6 families and 30 unrelated controls were performed by PCR RFLP analysis. Results\ Deletions of SMN gene detected in 9 of 10 (90%) cases of presumed SMA . No deletions of SMN in the telomere were found in the other members of families and controls.Conclusion\ PCR RFLP is a sensitive, specific and simple method in diagnosis of SMA.\;展开更多
Background Nontuberculous mycobacterium （NTM） had been reported to cause cutaneous infections which are difficult to interpret due to the variability of the clinical manifestations. Among NTM infections, Mycobacteri...Background Nontuberculous mycobacterium （NTM） had been reported to cause cutaneous infections which are difficult to interpret due to the variability of the clinical manifestations. Among NTM infections, Mycobacterium marinum （M. marinum） are mostly seen to cause skin infection. It is therefore important to establish a rapid approach for detection and identification of M. marinum from lesions of patients with suspected M. marinum infections. Methods Specimens were obtained from 5 patients with swimming pool granuloma. DNA was extracted and polymerase chain reaction （PCR） was performed. PCR products were digested with Hae III and BstE II, then analysed by pattern restriction analysis to detect heat shock protein （hsp） 65 kD gene. Results The 65 kD hsp gene was found in all specimens from patients with swimming pool granuloma. PCR restriction analysis （PRA） identified all 5 samples to be M. marinum infections, and the result was consistent with that of routine bacteriological identification. The lesions subsided or markedly improved upon treatment. Conclusions PRA is a sensitive, specific and rapid method in identification of mycobacteria. Application of this method will be helpful for early diagnosis of mycobacterial skin infections.展开更多
INTRODUCTIONLeprosy caused by Mvcobacterium leprae （M. leprae）, is a chronic granulomatous disease affecting the skin and peripheral nervous system, which is transmitted through direct contact with nontreated or ina...INTRODUCTIONLeprosy caused by Mvcobacterium leprae （M. leprae）, is a chronic granulomatous disease affecting the skin and peripheral nervous system, which is transmitted through direct contact with nontreated or inadequate treatment patients. Diagnosis of leprosy depends on the clinical signs and symptoms and slit skin smear positivity. However, it＇s sometimes similar with other granulomatous disease caused by mycobacterial infection. Early stage leprosy is difficult to diagnose by clinical criterion alone because the sensitivity of acid-fast bacilli staining is quite low. The polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） shows the great advantage in rapid identification and diagnosis for early cases and has a differentiation between leprosy and nonleprosy cases.展开更多
目的构建含有小鼠血清和糖皮质激素诱导蛋白激酶3(serum and glucocorticoid-induced protein kinase 3,SGK3)基因的真核表达载体pcDNA3.1-MYC-SGK3-mCherry,并观察和验证其在转染细胞HEK293中的表达。方法通过聚合酶链式反应将实验室...目的构建含有小鼠血清和糖皮质激素诱导蛋白激酶3(serum and glucocorticoid-induced protein kinase 3,SGK3)基因的真核表达载体pcDNA3.1-MYC-SGK3-mCherry,并观察和验证其在转染细胞HEK293中的表达。方法通过聚合酶链式反应将实验室保存的真核表达质粒pcDNA3.1-MYC-SGK3中目的基因SGK3与mCherry融合并扩增出来,然后定向克隆至pcDNA3.1-MYC质粒中,经限制性内切酶消化和测序证实后,通过脂质体法转染HEK293细胞,Western blotting法检测目的基因的蛋白表达情况。结果测序结果与之前预期结果相符,证实pcDNA3.1-MYC-SGK3-mCherry真核表达载体构建成功。Western blotting结果显示,转染pcDNA3.1-MYC-SGK3-mCherry的HEK293细胞出现清晰的阳性反应条带,说明目的片段成功表达。结论pcDNA3.1-MYC-SGK3-mCherry真核表达载体构建成功。展开更多
文摘Objective:To investigate the polyadenylation of mRNA in E. Coli. Methods: The mRNA of E. Coli was enriched from the total RNA with oligo(dT)-cellulose, prior to reverse transcription using oligo(dT)18as the primer. Double-stranded cDNA was subsequently synthesized, which was subjected to digestion with Sau3A I to produce multiple gene fragments for ligation with the adapters. PCR was carried out in 10 groups according to 10 different pairs of the selective primers, and the PCR products were then cloned into T-vectors. Results: More than 100 gene fragments had been cloned, 30 of which were sequenced. Conclusion:Polyadenylation of E. Coli mRNA may not be a biochemical curiosity but a general attribute of bacterial mRNA.
基金Supported by a National Research Foundation(NRF)of Korea grant funded by the Korean government(Ministry of EducationScience+2 种基金and TechnologyMEST)Grant No.2013-005810
文摘AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.
文摘Objective To understand the deletion in the survival motor neuron gene (SMN) of childhood onset spinal muscular atrophy (SMA) in Chinese, and the value of diagnosis of SMA using polymerase chain reaction restriction fragment length polymorphism (PCR RFLP)method. Methods\ Deletions of SMN gene of exon 7 and 8 in 10 cases of presumed SMA, and 20 normal controls from 6 families and 30 unrelated controls were performed by PCR RFLP analysis. Results\ Deletions of SMN gene detected in 9 of 10 (90%) cases of presumed SMA . No deletions of SMN in the telomere were found in the other members of families and controls.Conclusion\ PCR RFLP is a sensitive, specific and simple method in diagnosis of SMA.\;
文摘Background Nontuberculous mycobacterium （NTM） had been reported to cause cutaneous infections which are difficult to interpret due to the variability of the clinical manifestations. Among NTM infections, Mycobacterium marinum （M. marinum） are mostly seen to cause skin infection. It is therefore important to establish a rapid approach for detection and identification of M. marinum from lesions of patients with suspected M. marinum infections. Methods Specimens were obtained from 5 patients with swimming pool granuloma. DNA was extracted and polymerase chain reaction （PCR） was performed. PCR products were digested with Hae III and BstE II, then analysed by pattern restriction analysis to detect heat shock protein （hsp） 65 kD gene. Results The 65 kD hsp gene was found in all specimens from patients with swimming pool granuloma. PCR restriction analysis （PRA） identified all 5 samples to be M. marinum infections, and the result was consistent with that of routine bacteriological identification. The lesions subsided or markedly improved upon treatment. Conclusions PRA is a sensitive, specific and rapid method in identification of mycobacteria. Application of this method will be helpful for early diagnosis of mycobacterial skin infections.
基金The study was supported by a grant from the National Natural Science Foundation of China （No. 81271761）.
文摘INTRODUCTIONLeprosy caused by Mvcobacterium leprae （M. leprae）, is a chronic granulomatous disease affecting the skin and peripheral nervous system, which is transmitted through direct contact with nontreated or inadequate treatment patients. Diagnosis of leprosy depends on the clinical signs and symptoms and slit skin smear positivity. However, it＇s sometimes similar with other granulomatous disease caused by mycobacterial infection. Early stage leprosy is difficult to diagnose by clinical criterion alone because the sensitivity of acid-fast bacilli staining is quite low. The polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） shows the great advantage in rapid identification and diagnosis for early cases and has a differentiation between leprosy and nonleprosy cases.
文摘目的构建含有小鼠血清和糖皮质激素诱导蛋白激酶3(serum and glucocorticoid-induced protein kinase 3,SGK3)基因的真核表达载体pcDNA3.1-MYC-SGK3-mCherry,并观察和验证其在转染细胞HEK293中的表达。方法通过聚合酶链式反应将实验室保存的真核表达质粒pcDNA3.1-MYC-SGK3中目的基因SGK3与mCherry融合并扩增出来,然后定向克隆至pcDNA3.1-MYC质粒中,经限制性内切酶消化和测序证实后,通过脂质体法转染HEK293细胞,Western blotting法检测目的基因的蛋白表达情况。结果测序结果与之前预期结果相符,证实pcDNA3.1-MYC-SGK3-mCherry真核表达载体构建成功。Western blotting结果显示,转染pcDNA3.1-MYC-SGK3-mCherry的HEK293细胞出现清晰的阳性反应条带,说明目的片段成功表达。结论pcDNA3.1-MYC-SGK3-mCherry真核表达载体构建成功。
