The rRNA genetic locus is found in all prokaryotic organisms, and is highly conservative, although its relatively stable variations are found frequently in different bacteria. The utility of this locus as a taxonomic ...The rRNA genetic locus is found in all prokaryotic organisms, and is highly conservative, although its relatively stable variations are found frequently in different bacteria. The utility of this locus as a taxonomic and phylogenetic tool has been reported widely. This study, aimed at 16S rRNA gene (16S rDNA) and with the help of biomolecular methods, attempted to achieve the goal of rapid identification of common pathogens. In this study, 333 clinical isolated pathogenic bacteria were collected. Two pairs of primers were chosen and labeled with different fluorescent dyes and then used to amplify the genomic DNA extracted from bacteria. The PCR products were then detected by capillary electrophoresis-single strand conformation polymorphism (CE-SSCP). In order to pursue higher resolution and peak-separation effect, a high efficient separating medium, liner polyacrylamidedel (LPA), was put to use in this study. Finally, every bacteria colony generated distinct patterns from each other, which were easily to be used for identification. These results indicated that PCR-CE-SSCP was a rapid identification method for bacterial identification, with the aspects of high efficiency and high precision. Compared with traditional method, this technology is of great utility for clinical use especially for its high sensitivity.展开更多
BACKGROUND: Since single nucleotide polymorphisms (SNPs) can serve as gene markers, polymorphism profiles may help scientists to identify the full collection of genes that contribute to the development of complex dise...BACKGROUND: Since single nucleotide polymorphisms (SNPs) can serve as gene markers, polymorphism profiles may help scientists to identify the full collection of genes that contribute to the development of complex diseases such as cancer. The distribution of interleukin-10 (IL-10) promoter polymorphisms in Chinese Han ethnic patients with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) was investigated in this study. METHODS: The polymorphisms of IL-10 promoter region were detected by pulymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing. Sixty-six health controls, 42 patients with HBV infection, 30 HCC patients, and cell line SMMC-7721 were examined this way. RESULTS: Polyrnorphisms of T/C or T/N on-872 site occurred frequently in Han ethnic population. Pulyrnorphisms were detected in HBV and HCC patients and cell line SMMC-7721. The hotspot among the pulymorphisms was inserting base A between-1058 and-1057. CONCLUSION: Polymorphisms of IL-10 promoter in HBV and HCC patients may be associated with HBV infection and HCC development.展开更多
By employing different gel components and electrophoresis conditions, the PCR-SSCP(polymerase chain reaction-single-stranded conformation polymorphism) method was used to detect the polymorphism(CCC or CGC ) of codon ...By employing different gel components and electrophoresis conditions, the PCR-SSCP(polymerase chain reaction-single-stranded conformation polymorphism) method was used to detect the polymorphism(CCC or CGC ) of codon 72 in exon 4 of p53 gene. The results展开更多
Poly(ADP-ribose)polymerase-1(PARP-1)can exacerbate ischemic brain injury and lessen ischemic neuronal death,which may be associated with PARP-1 polymorphisms.The present study investigated human PARP-1 gene polymorphi...Poly(ADP-ribose)polymerase-1(PARP-1)can exacerbate ischemic brain injury and lessen ischemic neuronal death,which may be associated with PARP-1 polymorphisms.The present study investigated human PARP-1 gene polymorphisms in various Chinese nationalities,the results of which could potentially help in the treatment and prevention of neurologic diseases.Genetic polymorphisms of seven exons in the PARP-1 gene,in 898 Chinese Han,Buyi,Shui,Miao,and Zhuang subjects,were investigated by PCR-single-strand conformation polymorphism.A single-strand conformation polymorphism variant in exons 12,13,16,and 17 of the PARP-1 gene was identified in 148 people,with two stationary bands showing three degenerative single strands.Results showed that the PARP-1 gene polymorphisms exist in various nationalities,and may act as a biomarker for susceptibility to disease.展开更多
AIM:To investigate the relationship between c.343A>G and c.2216A>C polymorphism sites in the CDH17 gene and colorectal carcinoma.METHODS:Ninety-three non-consanguineous colorectal carcinoma patients admitted to ...AIM:To investigate the relationship between c.343A>G and c.2216A>C polymorphism sites in the CDH17 gene and colorectal carcinoma.METHODS:Ninety-three non-consanguineous colorectal carcinoma patients admitted to the Department of Oncology at the First Affiliated Hospital of Zhengzhou University were included in this study.Ninety-three peripheral venous blood samples,of approximately one milliliter from each patient,were collected betweenDecember 2009 and August 2010.The genomic DNA of these peripheral venous blood samples were extracted and purified using a Fermentas Genomic DNA Purification Kit(Fermentas,CA) according to the manufacturer' s protocol.The single nucleotide polymorphisms(SNPs) of the liver-intestine cadherin(CDH17) gene c.343A>G and c.2216A>C were determined by the polymerase chain reaction-single strand conformation polymorphism method(PCR-SSCP) in 93 peripheral venous blood samples from patients suffering with colorectal carcinoma.Typical samples that showed different migration bands in SSCP were confirmed by sequencing.Directed DNA sequencing was used to check the correctness of the genotype results from the PCR-SSCP method.RESULTS:There was a significant association between the c.2216 A>C SNPs of the CDH17 gene and the tumor-node-metastasis(TNM) grade,as well as with lymph node status,in 93 peripheral venous blood samples from colorectal carcinoma patients.The genotype frequencies of A/C,A/A,and C/C were 12.90%,33.33% and 53.76%,respectively.There was a significant correlation between lymph node metastasis,TNM grade,and the genotype distribution(P < 0.05).The C/C genotype raised the risk of lymph node metastasis and the TNM grade.There was a significant difference in the TNM grade and lymph node metastasis between the A/A and C/C genotypes(P = 0.003 and P = 0.013,respectively).Patients with colorectal carcinoma carrying the C allele tended to have a higher risk of lymph node metastasis and have a higher TNM grade.The difference between the TNM grades,as well as the lymph node metastasis of the two alleles,was statistically significant(P < 0.01).CONCLUSION:The SNPs of the CDH17 gene c.2216 A>C might be clinically important in the prognosis of colorectal carcinoma.展开更多
Objective: To study the relationship between the polymorphism of drug resistant gene rpoB and drug resistance against rifampicin(RFP) of M. tuberculosis L-forms, and to evaluate its clinical application. Methods: A to...Objective: To study the relationship between the polymorphism of drug resistant gene rpoB and drug resistance against rifampicin(RFP) of M. tuberculosis L-forms, and to evaluate its clinical application. Methods: A total of 52 clinical isolated strains of M. tuberculosis L-forms were collected. rpoB gene polymorphism was analyzed by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) and conventional antimicrobial susceptibility test (AST). Their results were compared. Results: AST results showed that 38 of 52 clinical isolated strains were drug resistance (73.08％),while PCR-SSCP indicated 65.38％ (32/52) rpoB gene polymorphism. There was no statistic significance(χ2= 2.4914) between the 2 methods. Conclusion:Combined the application of PCR-SSCP with AST in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis may have advantages at earlier diagnosis and guidance of clinical medications.展开更多
In order to investigate microbial community structures in different wastewater treatment processes and understand the relationship between the structures and the status of processes,the microbial community diversity,v...In order to investigate microbial community structures in different wastewater treatment processes and understand the relationship between the structures and the status of processes,the microbial community diversity,variety and distribution in five wastewater treatment pro cesses were studied by a culture-independent genetic fingerprinting technique single-strand conformation poly-morphism(SSCP).The five processes included denitrifying and phosphate-removal system(diminished N),Chinese traditional medicine wastewater treatment system(P),beer wastewater treatment system(W),fermentative biohydrogen-producing system(H),and sulfate-reduction system(S).The results indicated that the microbial community profiles in the wastewater bioreactors with the uniform status were very similar.The diversity of microbial populations was correlated with the complexity of organic contaminants in wastewater.Chinese traditional medicine wastewater contained more complex organic components;hence,the population diversity was higher than that of simple nutrient bioreactors fed with molasses wastewater.Compared with the strain bands in a simulated community,the relative proportion of some functional microbial populations in bioreactors was not dom-inant.Fermentative biohydrogen producer Ethanoligenens harbinense in the better condition bioreactor had only a 5% band density,and the Desulfovibrio sp.in the sulfate-reducing bioreactor had less than 1.5%band density.The SSCP profiles could identify the difference in microbial community structures in wastewater treatment processes,monitor some of the functional microbes in these processes,and consequently provide useful guidance for improving their efficiency.展开更多
Objective To develop a high throughput mutational detection method by mutiple fluorescence-labeled polymerase chain reaction(PCR)products.Methods A total of 27 known mutations including 22 substitutions,3 insertions(1...Objective To develop a high throughput mutational detection method by mutiple fluorescence-labeled polymerase chain reaction(PCR)products.Methods A total of 27 known mutations including 22 substitutions,3 insertions(1,2 and 7 bp)and 2 deletions(1 and 2 bp)in the hepatocyte nuclear factor(HNF)-4α,glucokinase and HNF-1α genes were tested.During nested PCR,amplified fragments were labeled with three fluorescent dyes.PCR products were visualized with an ABI-377 fluorescence sequencer using 5% glycerol or 10% sucrose in nondenaturing gel conditions.Results Twenty-five of 27 variants(93%)could be detected by combining 5% glycerol and 10% sucrosegel matrix conditions.Twenty-two of 27(82%)and 18 of 27(67%)variants were identified using 5%glycerol and 10% sucrose conditions,respectively.Conclusion This fluorescence-based PCR single strand conformation polymorphism technique represents a simple,non-hazardous,time-saving and sensitive method for high throughput mutation detection.展开更多
OBJECTIVE: To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. METHODS: The screenings were car...OBJECTIVE: To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. METHODS: The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells, and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. RESULTS: Two polymorphisms, C (-106) T and C (-12) G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C (-12) G and WT/C (-106) T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C (-106) T were 31.5% and 17.5% (P 0.05) respectively. The total frequency of WT/C (-12) G and WT/C (-106) T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P展开更多
OBJECTIVE: To clone and study the polymorphism within interleukin-4 (IL-4) proximal promoter of asthmatic children. METHODS: The IL-4 proximal promoter segments were amplified and selected by polymerase chain reaction...OBJECTIVE: To clone and study the polymorphism within interleukin-4 (IL-4) proximal promoter of asthmatic children. METHODS: The IL-4 proximal promoter segments were amplified and selected by polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) with genomic DNA from ten healthy children and forty patients with dominantly allergic familial histories as templates. The selected PCR segments were cloned into recombinant plasmids pIL-4-Jx2. The PCR inserts were sequenced by dideoxy chain termination method. RESULTS: Seven aberrant bands were found in SSCP analysis from forty asthmatic patients. The sequencing results showed that four variant sites were found within or adjacent to the known IL-4 regulatory element. A C to A transversion located at -229 position was just within the positive regulatory element-I (PRE-I) in one patient. A C to T transition adjacent to the negative regulatory element-II (NRE-II) and an extra G adjacent to TATA box were found in two patients. A five base nucleotide deletion was found near signal transducers and activators of transcription-6 responsive element (STAT-6 RE) in one patient. CONCLUSION: There were polymorphisms within the IL-4 proximal promoter of allergic asthmatic patients and these polymorphisms might result in aberrant expression of IL-4 gene and asthma.展开更多
Abstract Objective To establish a convenient method to detect the genomic population with hepatitis C virus (HCV) at nonstructure 5A (NS5A) region and to determine the correlation between the genomic population compl...Abstract Objective To establish a convenient method to detect the genomic population with hepatitis C virus (HCV) at nonstructure 5A (NS5A) region and to determine the correlation between the genomic population complexity at NS5A region and disease stage. Methods The sera from 52 patients with chronic hepatitis C virus infections were analysed using single strand conformation polymorphism (SSCP). In the SSCP, an asymmetric polymerase chain reaction (PCR) was carried out on the 455 bp products of the first round PCR at the NS5A region and the number of band of single strand deoxyribonucleic acid (DNA) which reacted with complemental DNA probe specific for the NS5A region after gel electrophoresis was analyzed. Results In 90% patients with chronic persistent hepatitis, the bands of single strand DNA was limited to one, and in those with chronic active hepatitis or liver cirrhosis, two or more bands of DNA were frequently detected. In about half of patients with hepatocellularcarcinoma, three or more bands were found. The number of bands increased with the progression of liver disease. The multivariate analysis showed that the progression of liver disease was the independent factor of viral diversity (P<0.025) and was not related to the age, sex, the route of infection and the titer of hepatitis C virus ribonucleic acid (HCV RNA). Conclusion These results suggest that the genomic variability of HCV at NS5A region increases with the progression of liver disease, and this may be closely related to the clinical features of type C liver disease.展开更多
AIM: In hepatocellular carcinoma (HCC) prevalent areas of China, the point mutation of p53 exon7 is highly correlated with Hepatitis B virus(HBV) infection and aflatoxin B intake. While in non-HCC-prevalent areas of C...AIM: In hepatocellular carcinoma (HCC) prevalent areas of China, the point mutation of p53 exon7 is highly correlated with Hepatitis B virus(HBV) infection and aflatoxin B intake. While in non-HCC-prevalent areas of China, these factors are not so important in the etiology of HCC. Therefore, the point mutation of p53 exon7 may also be different than that in HCC-prevalent areas of China. The aim of this study is to investigate the status and carcinogenic role of the point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province, a non-HCC-prevalent area in China. METHODS: PCR PCR-SSCP and PCR-RFLP were applied to analyze the homozygous deletion and point mutation of p53 exon7 in HCC samples from Anhui, which were confirmed by DNA sequencing and Genbank comparison. RESULTS: In the 38 samples of hepatocellular carcinoma, no homozygous deletion of p53 exon7 was detected and point mutations of p53 exon7 were found in 4 cases, which were found to be heterozygous mutation of codon 249 with a mutation rate of 10.53%(4/38). The third base mutation(G-T) of p53 codon 249 was found by DNA sequencing and Genbank comparison. CONCLUSION: The incidence of point mutation of p53 codon 249 is lower in hepatocellular carcinoma and the heterozygous mutation of p53 exon7 found in these patients only indicate that they have genetic susceptibility to HCC. p53 codon 249 is a hotspot of p53 exon7 point mutation, suggesting that the point mutation of p53 exon 7 may not play a major role in the carcinogenesis of HCC in Anhui Province, a non-HCC-prevalent area in China.展开更多
Objective：To study the genetic variation of mitochondrial DNA （mtDNA） among common laboratory strains of inbred mice. Methods： The genetic polymorphism of mtDNA among 4 classical laboratory strains of inbred mice ...Objective：To study the genetic variation of mitochondrial DNA （mtDNA） among common laboratory strains of inbred mice. Methods： The genetic polymorphism of mtDNA among 4 classical laboratory strains of inbred mice and 3 inbred strains of mice established in China was analyzed by polymerase chain reaction coupled with restriction fragment length polymorphism（PCR-RFLP） and PCR coupled with single-stranded conformation polymorphism（PCR-SSCP）. Results： With regard to the D-loop （Displacement loop, D-loop）, tRNA^ Met＋Glu＋Ile, and ND3 （NADH dehydrogenase subunit 3, ND3） gene fragments of mtDNA from these mice,no variation was revealed by PCR-RFLP at 46 restriction enzyme sites. Further analyzed by PCR-SSCP,the D-loop 5＇fragment and 3＇end fragment of mtDNA from these mice also showed no genetic variation. Conclusion： Owing to maternal mode of inheritance of mtDNA,the results indicate that these common inbred strains of mice share the same maternal lineage.展开更多
AIM: To explore the virulence and the potential pathogenicity of coccoid Helicobacter pylori (H. pylori) transformed from spiral form by exposure to antibiotic. METHODS: Three strains of H. pylori, isolated from gastr...AIM: To explore the virulence and the potential pathogenicity of coccoid Helicobacter pylori (H. pylori) transformed from spiral form by exposure to antibiotic. METHODS: Three strains of H. pylori, isolated from gastric biopsy specimens of confirmed peptic ulcer, were converted from spiral into coccoid from by exposure to metronidazole. Both spiral and coccoid form of H. pylori were tested for the urease activity, the adherence to Hep-2 cells and the vacuolating cytotoxicity to Hela cells, and the differences of the protein were analysed by SDS-PAGE and Western blot. The mutation of the genes including ureA, ureB,hpaA, vacA and cagA, related with virulence, was detected by means of PCR and PCR-SSCP. RESULTS: In the coccoid H. pylori,the urease activity, the adherence to Hep-2 cells and the vacuolating cytotoxicity to Hela cells all decreased. In strain F44, the rate and index of adherence reduced from 70.0% +/- 5.3% to 33% +/- 5.1% and from 2.6 +/- 0.4 to 0.96 +/- 0.3 (P 【 0.01), respectively. The invasion of coccoid H. pylori into Hep-2 cell could be seen under electronmicroscope. SDS-PAGE showed that the content of the protein with the molecular weight over Mr 74000 decreased, and the hybriditional signal in band M(r) 125000 weakened, while the band M(r)110000 and M(r)63000 strengthened in coccoid H.pylori as shown in Western blot. The results of PCR were all positive, and PCR-SSCP indicated that there may exist the point mutation in gene hpaA or vacA. CONCLUSION: The virulence and the proteins with molecular weight over M(r)74000 in coccoid H.pylori decrease, but no deletion exists in amplification fragments from ureA, ureB, hpaA, vacA and cagA genes, suggesting that coccoid H.pylori may have potential pathogenicity.展开更多
Objective To explore the molecular mechanism of insulin resistance in the patients with polycystic ovarian syndrome (PCOS)Methods Polymerase chain reaction, silver staining-single strand conformation poly-morphism(PCR...Objective To explore the molecular mechanism of insulin resistance in the patients with polycystic ovarian syndrome (PCOS)Methods Polymerase chain reaction, silver staining-single strand conformation poly-morphism(PCR-SSCP) and DNA direct sequencing were used to detect the mutation of insulin receptor (INSR) gene in exon 17-21 with the abdominal wall adipose tissue from 31 patients with PCOS (PCOS Group) and 30 patients with pure hysteromyoma in reproductive lift (Control Group).Results Tiventy-two variant SSCP patterns in exon 17 of INSR gene were detected. Direct sequence analysis of exon 17 showed that homozygous nonsense mutation was two alleles single nucleotide polymorphism (SNP) at the codon 1058 (CAC→CAT). Exons 18-21were not detected with any significantly mutation. The INSR gene His1058C→ T substitution collecting rate and insulin resistance were significantly higher in the PCOS group than in the control group (P = 0. 0293, P<0. 05, P<0. 01). Conclusion It is suggested that the SNP in codon 1058 of the INSR gene might be related with the insulin resistance in PCOS patients, which has hereditary tendency. And the missense mutation,nonsense mutation and frameshift mutation at exons 18-21 in tyrosine protein kinase region of INSR gene for PCOS patients were not frequently observed.展开更多
文摘The rRNA genetic locus is found in all prokaryotic organisms, and is highly conservative, although its relatively stable variations are found frequently in different bacteria. The utility of this locus as a taxonomic and phylogenetic tool has been reported widely. This study, aimed at 16S rRNA gene (16S rDNA) and with the help of biomolecular methods, attempted to achieve the goal of rapid identification of common pathogens. In this study, 333 clinical isolated pathogenic bacteria were collected. Two pairs of primers were chosen and labeled with different fluorescent dyes and then used to amplify the genomic DNA extracted from bacteria. The PCR products were then detected by capillary electrophoresis-single strand conformation polymorphism (CE-SSCP). In order to pursue higher resolution and peak-separation effect, a high efficient separating medium, liner polyacrylamidedel (LPA), was put to use in this study. Finally, every bacteria colony generated distinct patterns from each other, which were easily to be used for identification. These results indicated that PCR-CE-SSCP was a rapid identification method for bacterial identification, with the aspects of high efficiency and high precision. Compared with traditional method, this technology is of great utility for clinical use especially for its high sensitivity.
文摘BACKGROUND: Since single nucleotide polymorphisms (SNPs) can serve as gene markers, polymorphism profiles may help scientists to identify the full collection of genes that contribute to the development of complex diseases such as cancer. The distribution of interleukin-10 (IL-10) promoter polymorphisms in Chinese Han ethnic patients with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) was investigated in this study. METHODS: The polymorphisms of IL-10 promoter region were detected by pulymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing. Sixty-six health controls, 42 patients with HBV infection, 30 HCC patients, and cell line SMMC-7721 were examined this way. RESULTS: Polyrnorphisms of T/C or T/N on-872 site occurred frequently in Han ethnic population. Pulyrnorphisms were detected in HBV and HCC patients and cell line SMMC-7721. The hotspot among the pulymorphisms was inserting base A between-1058 and-1057. CONCLUSION: Polymorphisms of IL-10 promoter in HBV and HCC patients may be associated with HBV infection and HCC development.
文摘By employing different gel components and electrophoresis conditions, the PCR-SSCP(polymerase chain reaction-single-stranded conformation polymorphism) method was used to detect the polymorphism(CCC or CGC ) of codon 72 in exon 4 of p53 gene. The results
基金the National Natural Science Foundation of China,No.30972500the Natural Science Foundation of Guangdong Province,No.7301507
文摘Poly(ADP-ribose)polymerase-1(PARP-1)can exacerbate ischemic brain injury and lessen ischemic neuronal death,which may be associated with PARP-1 polymorphisms.The present study investigated human PARP-1 gene polymorphisms in various Chinese nationalities,the results of which could potentially help in the treatment and prevention of neurologic diseases.Genetic polymorphisms of seven exons in the PARP-1 gene,in 898 Chinese Han,Buyi,Shui,Miao,and Zhuang subjects,were investigated by PCR-single-strand conformation polymorphism.A single-strand conformation polymorphism variant in exons 12,13,16,and 17 of the PARP-1 gene was identified in 148 people,with two stationary bands showing three degenerative single strands.Results showed that the PARP-1 gene polymorphisms exist in various nationalities,and may act as a biomarker for susceptibility to disease.
基金Supported by 2010A310011 Henan Provincial Department of Education on Natural Science Research Projects
文摘AIM:To investigate the relationship between c.343A>G and c.2216A>C polymorphism sites in the CDH17 gene and colorectal carcinoma.METHODS:Ninety-three non-consanguineous colorectal carcinoma patients admitted to the Department of Oncology at the First Affiliated Hospital of Zhengzhou University were included in this study.Ninety-three peripheral venous blood samples,of approximately one milliliter from each patient,were collected betweenDecember 2009 and August 2010.The genomic DNA of these peripheral venous blood samples were extracted and purified using a Fermentas Genomic DNA Purification Kit(Fermentas,CA) according to the manufacturer' s protocol.The single nucleotide polymorphisms(SNPs) of the liver-intestine cadherin(CDH17) gene c.343A>G and c.2216A>C were determined by the polymerase chain reaction-single strand conformation polymorphism method(PCR-SSCP) in 93 peripheral venous blood samples from patients suffering with colorectal carcinoma.Typical samples that showed different migration bands in SSCP were confirmed by sequencing.Directed DNA sequencing was used to check the correctness of the genotype results from the PCR-SSCP method.RESULTS:There was a significant association between the c.2216 A>C SNPs of the CDH17 gene and the tumor-node-metastasis(TNM) grade,as well as with lymph node status,in 93 peripheral venous blood samples from colorectal carcinoma patients.The genotype frequencies of A/C,A/A,and C/C were 12.90%,33.33% and 53.76%,respectively.There was a significant correlation between lymph node metastasis,TNM grade,and the genotype distribution(P < 0.05).The C/C genotype raised the risk of lymph node metastasis and the TNM grade.There was a significant difference in the TNM grade and lymph node metastasis between the A/A and C/C genotypes(P = 0.003 and P = 0.013,respectively).Patients with colorectal carcinoma carrying the C allele tended to have a higher risk of lymph node metastasis and have a higher TNM grade.The difference between the TNM grades,as well as the lymph node metastasis of the two alleles,was statistically significant(P < 0.01).CONCLUSION:The SNPs of the CDH17 gene c.2216 A>C might be clinically important in the prognosis of colorectal carcinoma.
文摘Objective: To study the relationship between the polymorphism of drug resistant gene rpoB and drug resistance against rifampicin(RFP) of M. tuberculosis L-forms, and to evaluate its clinical application. Methods: A total of 52 clinical isolated strains of M. tuberculosis L-forms were collected. rpoB gene polymorphism was analyzed by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) and conventional antimicrobial susceptibility test (AST). Their results were compared. Results: AST results showed that 38 of 52 clinical isolated strains were drug resistance (73.08％),while PCR-SSCP indicated 65.38％ (32/52) rpoB gene polymorphism. There was no statistic significance(χ2= 2.4914) between the 2 methods. Conclusion:Combined the application of PCR-SSCP with AST in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis may have advantages at earlier diagnosis and guidance of clinical medications.
基金This work was supported by the National Natural Science Foundation of China(Grant Nos.:50208006,30470054 and 50678049)China Postdoctoral Science Foundation(Grant No.:20070410266).
文摘In order to investigate microbial community structures in different wastewater treatment processes and understand the relationship between the structures and the status of processes,the microbial community diversity,variety and distribution in five wastewater treatment pro cesses were studied by a culture-independent genetic fingerprinting technique single-strand conformation poly-morphism(SSCP).The five processes included denitrifying and phosphate-removal system(diminished N),Chinese traditional medicine wastewater treatment system(P),beer wastewater treatment system(W),fermentative biohydrogen-producing system(H),and sulfate-reduction system(S).The results indicated that the microbial community profiles in the wastewater bioreactors with the uniform status were very similar.The diversity of microbial populations was correlated with the complexity of organic contaminants in wastewater.Chinese traditional medicine wastewater contained more complex organic components;hence,the population diversity was higher than that of simple nutrient bioreactors fed with molasses wastewater.Compared with the strain bands in a simulated community,the relative proportion of some functional microbial populations in bioreactors was not dom-inant.Fermentative biohydrogen producer Ethanoligenens harbinense in the better condition bioreactor had only a 5% band density,and the Desulfovibrio sp.in the sulfate-reducing bioreactor had less than 1.5%band density.The SSCP profiles could identify the difference in microbial community structures in wastewater treatment processes,monitor some of the functional microbes in these processes,and consequently provide useful guidance for improving their efficiency.
文摘Objective To develop a high throughput mutational detection method by mutiple fluorescence-labeled polymerase chain reaction(PCR)products.Methods A total of 27 known mutations including 22 substitutions,3 insertions(1,2 and 7 bp)and 2 deletions(1 and 2 bp)in the hepatocyte nuclear factor(HNF)-4α,glucokinase and HNF-1α genes were tested.During nested PCR,amplified fragments were labeled with three fluorescent dyes.PCR products were visualized with an ABI-377 fluorescence sequencer using 5% glycerol or 10% sucrose in nondenaturing gel conditions.Results Twenty-five of 27 variants(93%)could be detected by combining 5% glycerol and 10% sucrosegel matrix conditions.Twenty-two of 27(82%)and 18 of 27(67%)variants were identified using 5%glycerol and 10% sucrose conditions,respectively.Conclusion This fluorescence-based PCR single strand conformation polymorphism technique represents a simple,non-hazardous,time-saving and sensitive method for high throughput mutation detection.
文摘OBJECTIVE: To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. METHODS: The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells, and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. RESULTS: Two polymorphisms, C (-106) T and C (-12) G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C (-12) G and WT/C (-106) T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C (-106) T were 31.5% and 17.5% (P 0.05) respectively. The total frequency of WT/C (-12) G and WT/C (-106) T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P
文摘OBJECTIVE: To clone and study the polymorphism within interleukin-4 (IL-4) proximal promoter of asthmatic children. METHODS: The IL-4 proximal promoter segments were amplified and selected by polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) with genomic DNA from ten healthy children and forty patients with dominantly allergic familial histories as templates. The selected PCR segments were cloned into recombinant plasmids pIL-4-Jx2. The PCR inserts were sequenced by dideoxy chain termination method. RESULTS: Seven aberrant bands were found in SSCP analysis from forty asthmatic patients. The sequencing results showed that four variant sites were found within or adjacent to the known IL-4 regulatory element. A C to A transversion located at -229 position was just within the positive regulatory element-I (PRE-I) in one patient. A C to T transition adjacent to the negative regulatory element-II (NRE-II) and an extra G adjacent to TATA box were found in two patients. A five base nucleotide deletion was found near signal transducers and activators of transcription-6 responsive element (STAT-6 RE) in one patient. CONCLUSION: There were polymorphisms within the IL-4 proximal promoter of allergic asthmatic patients and these polymorphisms might result in aberrant expression of IL-4 gene and asthma.
文摘Abstract Objective To establish a convenient method to detect the genomic population with hepatitis C virus (HCV) at nonstructure 5A (NS5A) region and to determine the correlation between the genomic population complexity at NS5A region and disease stage. Methods The sera from 52 patients with chronic hepatitis C virus infections were analysed using single strand conformation polymorphism (SSCP). In the SSCP, an asymmetric polymerase chain reaction (PCR) was carried out on the 455 bp products of the first round PCR at the NS5A region and the number of band of single strand deoxyribonucleic acid (DNA) which reacted with complemental DNA probe specific for the NS5A region after gel electrophoresis was analyzed. Results In 90% patients with chronic persistent hepatitis, the bands of single strand DNA was limited to one, and in those with chronic active hepatitis or liver cirrhosis, two or more bands of DNA were frequently detected. In about half of patients with hepatocellularcarcinoma, three or more bands were found. The number of bands increased with the progression of liver disease. The multivariate analysis showed that the progression of liver disease was the independent factor of viral diversity (P<0.025) and was not related to the age, sex, the route of infection and the titer of hepatitis C virus ribonucleic acid (HCV RNA). Conclusion These results suggest that the genomic variability of HCV at NS5A region increases with the progression of liver disease, and this may be closely related to the clinical features of type C liver disease.
基金the Natural Science Foundation of Anhui Province,No.99044312(WY) and No.9741006(LX)Natural Science Foundation of Anhui Educational Commission,No.JL-97-077(WY).
文摘AIM: In hepatocellular carcinoma (HCC) prevalent areas of China, the point mutation of p53 exon7 is highly correlated with Hepatitis B virus(HBV) infection and aflatoxin B intake. While in non-HCC-prevalent areas of China, these factors are not so important in the etiology of HCC. Therefore, the point mutation of p53 exon7 may also be different than that in HCC-prevalent areas of China. The aim of this study is to investigate the status and carcinogenic role of the point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province, a non-HCC-prevalent area in China. METHODS: PCR PCR-SSCP and PCR-RFLP were applied to analyze the homozygous deletion and point mutation of p53 exon7 in HCC samples from Anhui, which were confirmed by DNA sequencing and Genbank comparison. RESULTS: In the 38 samples of hepatocellular carcinoma, no homozygous deletion of p53 exon7 was detected and point mutations of p53 exon7 were found in 4 cases, which were found to be heterozygous mutation of codon 249 with a mutation rate of 10.53%(4/38). The third base mutation(G-T) of p53 codon 249 was found by DNA sequencing and Genbank comparison. CONCLUSION: The incidence of point mutation of p53 codon 249 is lower in hepatocellular carcinoma and the heterozygous mutation of p53 exon7 found in these patients only indicate that they have genetic susceptibility to HCC. p53 codon 249 is a hotspot of p53 exon7 point mutation, suggesting that the point mutation of p53 exon 7 may not play a major role in the carcinogenesis of HCC in Anhui Province, a non-HCC-prevalent area in China.
基金Supported by Developmental Programming Item of National Keystone Basic Research (G2000016106) and National Natural Science Foundation of China (No. 39600079)
文摘Objective：To study the genetic variation of mitochondrial DNA （mtDNA） among common laboratory strains of inbred mice. Methods： The genetic polymorphism of mtDNA among 4 classical laboratory strains of inbred mice and 3 inbred strains of mice established in China was analyzed by polymerase chain reaction coupled with restriction fragment length polymorphism（PCR-RFLP） and PCR coupled with single-stranded conformation polymorphism（PCR-SSCP）. Results： With regard to the D-loop （Displacement loop, D-loop）, tRNA^ Met＋Glu＋Ile, and ND3 （NADH dehydrogenase subunit 3, ND3） gene fragments of mtDNA from these mice,no variation was revealed by PCR-RFLP at 46 restriction enzyme sites. Further analyzed by PCR-SSCP,the D-loop 5＇fragment and 3＇end fragment of mtDNA from these mice also showed no genetic variation. Conclusion： Owing to maternal mode of inheritance of mtDNA,the results indicate that these common inbred strains of mice share the same maternal lineage.
基金Supported by the Natural Science Foundation of Fujian Province,China,No.95A003
文摘AIM: To explore the virulence and the potential pathogenicity of coccoid Helicobacter pylori (H. pylori) transformed from spiral form by exposure to antibiotic. METHODS: Three strains of H. pylori, isolated from gastric biopsy specimens of confirmed peptic ulcer, were converted from spiral into coccoid from by exposure to metronidazole. Both spiral and coccoid form of H. pylori were tested for the urease activity, the adherence to Hep-2 cells and the vacuolating cytotoxicity to Hela cells, and the differences of the protein were analysed by SDS-PAGE and Western blot. The mutation of the genes including ureA, ureB,hpaA, vacA and cagA, related with virulence, was detected by means of PCR and PCR-SSCP. RESULTS: In the coccoid H. pylori,the urease activity, the adherence to Hep-2 cells and the vacuolating cytotoxicity to Hela cells all decreased. In strain F44, the rate and index of adherence reduced from 70.0% +/- 5.3% to 33% +/- 5.1% and from 2.6 +/- 0.4 to 0.96 +/- 0.3 (P 【 0.01), respectively. The invasion of coccoid H. pylori into Hep-2 cell could be seen under electronmicroscope. SDS-PAGE showed that the content of the protein with the molecular weight over Mr 74000 decreased, and the hybriditional signal in band M(r) 125000 weakened, while the band M(r)110000 and M(r)63000 strengthened in coccoid H.pylori as shown in Western blot. The results of PCR were all positive, and PCR-SSCP indicated that there may exist the point mutation in gene hpaA or vacA. CONCLUSION: The virulence and the proteins with molecular weight over M(r)74000 in coccoid H.pylori decrease, but no deletion exists in amplification fragments from ureA, ureB, hpaA, vacA and cagA genes, suggesting that coccoid H.pylori may have potential pathogenicity.
基金This study was support by the National Nature Science Fund,P.R.China(30100200)
文摘Objective To explore the molecular mechanism of insulin resistance in the patients with polycystic ovarian syndrome (PCOS)Methods Polymerase chain reaction, silver staining-single strand conformation poly-morphism(PCR-SSCP) and DNA direct sequencing were used to detect the mutation of insulin receptor (INSR) gene in exon 17-21 with the abdominal wall adipose tissue from 31 patients with PCOS (PCOS Group) and 30 patients with pure hysteromyoma in reproductive lift (Control Group).Results Tiventy-two variant SSCP patterns in exon 17 of INSR gene were detected. Direct sequence analysis of exon 17 showed that homozygous nonsense mutation was two alleles single nucleotide polymorphism (SNP) at the codon 1058 (CAC→CAT). Exons 18-21were not detected with any significantly mutation. The INSR gene His1058C→ T substitution collecting rate and insulin resistance were significantly higher in the PCOS group than in the control group (P = 0. 0293, P<0. 05, P<0. 01). Conclusion It is suggested that the SNP in codon 1058 of the INSR gene might be related with the insulin resistance in PCOS patients, which has hereditary tendency. And the missense mutation,nonsense mutation and frameshift mutation at exons 18-21 in tyrosine protein kinase region of INSR gene for PCOS patients were not frequently observed.
