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Results for “Overlapping PCR”

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At least 271 records · Page 15Linked to original sources
PubMed2000

[Construction of membrane-anchored interleukin-6 receptor beta subunit mutant and its effect on IL-6 signal transduction].

OBJECTIVE: To investigate whether the membrane-anchored gp130 mutant containing the second and the third fibronectin type III modules of the extracellular region could still transduce IL-6 signal. METHOD: The membrane-anchored gp130 mutant cDNA was amplified by the overlap extension PCR and inserted into the mammalian expression vector pRc/RSV. The mutant was proved to be effectively expressed in SKO-007 cells and Jurkat cells by dot blot hybridization. The mutant's effects on IL-6 signaling in these two kinds of cells were detected by electrophoresis mobility shift assay (EMSA). RESULTS: In SKO-007 cells, the mutant could enhance the activated APRF, but the effect was not as strong as that of wide-type gp130. IL-6 could not activate nuclear factors in Jurkat cells. After the expression vectors pRc/RSVgp130 and pRc/RSV were transfected into Jurkat cells, IL-6 could activate APRF, but could not do NF-IL6. Similar to the results in SKO-007 cells, the effects of the mutant was also weaker than that of wild-type gp130. CONCLUSION: Although the mutant could still transduce IL-6 signal, it is not as efficiently as the wild-type gp130. It suggested that the Ig-like domain of gp130 also played an important role in IL-6 signal transduction.

Antigens, CD↗
PubMed2002

Mature insulin production by engineered non-beta cells.

OBJECTIVE: To pursue insulin and islet-transplantation replacement therapy for type 1 diabetes based on engineered human non-beta cells which secrete mature insulin. METHODS: Human proinsulin cDNA was cloned from its genomic gene and mutated by overlap extension PCR, introducing furin consensus cleavage sequences (Arg-Xaa-Lys/Arg-Arg). An expression vector encoding a genetically modified human proinsulin cDNA was generated and transduced to Hela, 293, and L02 cells by lipofectin-mediated DNA transfection. Following G418 screening, the surviving L02 cells were selected and enriched. Insulin levels in the supernatant and cells were evaluated using radioimmunoassay and immunofluorescence staining. RESULTS: Three sites in the insulin gene were mutated simultaneously. Insulin gene modified cells were able to express insulin at different levels: 8.45 - 188.00 microIU/24 h/2.0 x 10(6) Hela cells and 159.88 - 242.14 microIU/24 h/2.0 x 10(6) 293 cells for transient expression, and 2.56 - 61.95 microIU/24 h/2.0 x 10(6) from several L02 clones screened with G418. No insulin was released by control cells. Furthermore, immunofluorescence staining confirmed that proinsulin was stored as vacuoles in the cytoplasm of L02 cells. CONCLUSION: A correctly mutated human proinsulin cDNA was obtained successfully, transfected and expressed efficiently in non-beta cells, lending support to the study of somatic gene therapy in diabetes mellitus.

Cell Line↗
PubMed1999

[Study of the 3'noncoding region of Chinese hepatitis C virus genome].

OBJECTIVE: To analyze the 3' noncoding region (3' NCR) of HCV genome from Chinese hepatitis C patients so as to facilitate further study of mechanism of HCV gene replication. METHODS: Two different strategies were employed to amplify the full-length of the 3' noncoding region of HCV genome from sera of HCV infected patients in Shanghai area: one was to amplify the full-length fragment directly by nested PCR and the other amplify two overlapping fragments. The PCR products were further analyzed by sequencing and nucleotide alignments. A HCV genome 3'NCR based RT-PCR was developed and its specificity and sensitivity for HCV RNA detection in sera was compared with the established 5'NCR based RT-PCR. RESULTS: Sequence analysis showed that Chinese HCV genomic 3' NCR consists of three parts: the 5' region, poly (U-UC) tract and the 98-base region. Sequence alignments revealed that, while the 98-base regions were completely conserved in different isolates and were identical to the reported sequences, the poly (U-UC) region shared highly diversities. A high degree of concordance(95%) between the 3'NCR and 5'NCR RT-PCR for detection of HCV RNA in sera was found. CONCLUSION: The high conservation at the 3' NCR(98 bases) of HCV genome among different isolates indicated that this region may be critical for HCV gene replication The 3'NCR based RT-PCR may be a useful addition to available systems to diagnosis HCV infection.

3' Untranslated Regions↗
PubMed2005

[Expression, identification and bioactivity characterization of an anti-CD3/anti-CD20 bispecific single-chain antibody].

The synthetic gene with 1640bp encoding for the anti-CD3/anti-CD20 bispecific single-chain antibody was designed and obtained by SOE (splicing by overlap extension) PCR. The cDNA was cloned into Flp-In expression vector pcDNA5/FRT and transfeced into Flp-In CHO cells to generate a stable expression cell line with a capacity for expressing anti-CD3/anti-CD20 bispecific single-chain antibody at 300 microg/L. The protein, which had a molecular weight of about 70 kD,was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western-blot analysis. Immunofluorescence assay and cellular rosetting showed that it can react specifically on Jurkat (CD3+) and Ramous (CD20+) cells. The lysis of human PBL against CD20-positive lymphoma Ramous cells in the presence of the anti-CD3/anti-CD20 bispecific single-chain antibody can observed by microscope. All these results would lighten the further study of its biological functions in vitro and in vivo.

Animals↗
PubMed2006

[The effect of CACNA1H gene G773D mutation on calcium channel function].

OBJECTIVE: To study the effect of CACNA1H gene mutation G773D on calcium channel function. METHODS: By the overlap extension PCR we introduced G773D mutation into a human Cav3.2acDNA for constructing the mutant. And then using whole cell clamp technique, we studied the alterations of channel behavior in transfected HEK-293 cells. RESULTS: There were no difference in kinetics of activation and inactivation of calcium channel between wild type and mutant. However comparing with the wild-type Cav3.2 channel, G773D mutant could increase the calcium current density significantly. CONCLUSION: CACNA1H gene G773D mutation is able to increase calcium current and neuronal excitability.

Base Sequence↗
PubMed2006

[Gene construction, expression and activities analysis of human leptin].

Six 89bp primers were designed on the base of the cDNA sequence encoding the human leptin reported on the NCBI. The synthetic gene with 464bp encoding rhLep was obtained by SOE ( splicing by overlap extension) PCR. The expression vector pET22b(+)/rhLep was constructed and transformed into E. coli BL21 (DE3). The rhLep protein was expressed as inclusion bodies with the yield of more than 50% of total bacterial proteins after IPTG induction. The rhLep protein, which has a molecular weight about 16kD, was purified by Ni2+ affinity chromatography column and identified by SDS-PAGE. The MTT Assay shows that rhLep promotes EC304 cells growth at the low concentration of 10ng/mL to 30 ng/mL, and rhLep appears cytotoxic to EC304 cells with the high dose of 50ng/mL to 225ng/mL. The viability of EC304 cells decreases to 1.2% with the concentration of 225ng/mL of rhLep. The massive apoptosis of rhLep on EC304 cells is observed by AO-staining under fluorescent microscope. All these results would lay the foundation for the further study of its biological functions in vitro and in vivo.

Apoptosis↗
PubMed1992

NGF/BDNF chimeric proteins: analysis of neurotrophin specificity by homolog-scanning mutagenesis.

Despite their extensive sequence identities at the amino acid level (approximately 55%), NGF and brain-derived neurotrophic factor (BDNF) display distinct neuronal specificity toward neurons of both the PNS and CNS. To explore which region(s) within these neurotrophic factors might determine their differential actions on various subpopulations of peripheral neurons, a systematic series (homolog-scanning mutagenesis) of chimeric NGF/BDNF molecules was prepared using PCR overlap-extension techniques. After expression in COS-7 cells, the chimeric proteins were tested for their biological activities in neurite outgrowth and neuronal survival assays. This approach led to the functional expression of 12 NGF/BDNF chimeras. Surprisingly, despite replacing successive amino acid segments throughout the entire length of NGF with the corresponding parts of BDNF, all chimeras displayed full NGF-like activity in bioassays carried out with PC12 cells, embryonic chick dorsal root ganglion explants, sympathetic ganglion explants, and dissociated cultures of dorsal root ganglion neurons. Most of the chimeras additionally showed BDNF-like activity as defined by neurite outgrowth on chick nodose ganglion explants. However, none of the chimeras supported the survival of dissociated nodose ganglion neurons. Our results suggest that NGF and BDNF must share very similar higher-order protein structures, and we propose that the overall structure or conformation of NGF, in contrast to short amino acid "active-site" segments, may determine its exact neuronal specificity.

Amino Acid Sequence↗
PubMed1997

BRCA1 sequence variations in 160 individuals referred to a breast/ovarian family cancer clinic. Institut Curie Breast Cancer Group.

An account of familial aggregation in breast/ovarian cancer has become possible with the identification of BRCA1 germ-line mutations. We evaluated, for 249 individuals registered with the Institut Curie in Paris, the prior probability that an individual carried a mutation that predisposes to these diseases. We chose 160 women for BRCA1 analysis: 103 with a family history of breast cancer and 57 with a family history of breast-ovarian cancer. To detect small mutations, we generated and analyzed 35 overlapping genomic PCR products that cover the coding portion of the gene, by using denaturing gradient gel electrophoresis. Thirty-eight truncating mutations (32 frameshifts, 4 nonsense mutations, and 2 splice variants) were observed in 15% of women with a family history of breast cancer only and in 40% of those with a history of breast-ovarian cancer. Twelve of 25 distinct truncating mutations identified were novel and unique. Most BRCA1 mutations that had been reported more than five times in the Breast Cancer Information Core were present in our series. One mutation (5149del4) observed in two apparently unrelated families most likely originates from a common ancestor. The position of truncating mutations did not significantly affect the ratio of the risk of breast cancer to that of ovarian cancer. In addition, 15 DNA variants (14 missense mutations and 1 neutral mutation) were identified, 9 of which were novel. Indirect evidence suggests that seven of these mutations are deleterious.

Africa↗
PubMed1997

The construction and expression of deletion mutant of human pro-urokinase cDNA.

The human pro-urokinase mutant deleting 150-156 amino acids was constructed by overlap-extension PCR and other molecular cloning techniques. The mutant was expressed transiently in COS-7 cells and constitutively in CHO cells; the expression level is 450-500 IU/10(6) cells/24 h. SDS-PAGE and Western blotting analysis showed that the molecular weight of the expression product is 54 kDa, which is similar to that of the mature pro-UK and recombinant pro-UK (rPro-UK) expressed by full-length cDNA. Most of the products exist in the form of single chain; the percentage of single chain is much higher than that of rPro-UK. In addition, the mutant product is more resistant to proteinases and its affinity to fibrin is also improved slightly.

Animals↗
PubMed2000

HPV-16 E2 gene disruption and sequence variation in CIN 3 lesions and invasive squamous cell carcinomas of the cervix: relation to numerical chromosome abnormalities.

AIM: To test the hypothesis that, because the human papillomavirus (HPV) E2 protein represses viral early gene transcription, E2 gene sequence variation or disruption could play a part in the induction of the numerical chromosome abnormalities that have been described in squamous cervical lesions. METHODS: The integrity and sequence of the E2 gene from 11 cervical intraepithelial neoplasia (CIN) grade 3 lesions and 14 invasive squamous cell carcinomas, all of which contained HPV-16, were analysed by the polymerase chain reaction (PCR). The E2 gene was amplified in three overlapping fragments and PCR products sequenced directly. Chromosome abnormalities were identified by interphase cytogenetics using chromosome specific probes for chromosomes 1, 3, 11, 17, 18, and X. RESULTS: E2 gene disruption was present in significantly more invasive carcinomas (eight of 14) than CIN 3 lesions (one of 11) (p = 0.03). No association was found between E2 disruption and the presence of a numerical chromosome abnormality. The E2 gene from the non-disrupted isolates was sequenced and wild-type (n = 5) and variant (n = 11) sequences identified. Variant sequences belonged to European and African classes and contained from one to 15 amino acid substitutions. Although numerical chromosome abnormalities were significantly more frequent in invasive squamous cell carcinoma than CIN 3 (p = 0.04), there was no significant relation between the presence of sequence variation and either histological diagnosis or chromosome abnormality. CONCLUSIONS: These data do not support the hypothesis that E2 gene disruption or variation is important in the induction of chromosome imbalance in these lesions. However, there is a relation between E2 gene disruption and the presence of invasive disease.

Amino Acid Sequence↗
PubMed2005

[Construction and expression of anti-human AFP ScFv gene in BL-21 (DE3) E. coli].

OBJECTIVE: To construct anti-human AFP single chain fragment variable (ScFv) gene, transform it into BL-21 (DE3) E. coli for expression, and identify its bioactivity. METHODS: VH and VL genes of anti-human AFP monoclonal antibody were cloned by RT-PCR from hybridoma. The ScFv gene was spliced by sequence overlap extending (SOE) PCR, and then it was ligated into pGEM-T vector to be identified by endonuclease digestion, PCR and sequencing. ScFv gene was cloned into pET32 (a+) vector and transformed into BL-21 (DE3) E. coli. The positive clones were screened out by IPTG induction, and the ScFv antibody was purified to be identified by SDS-PAGE and competitive inhibition ELISA test. RESULTS: The VH DNA consisted of 339 bases, coming from the mouse IgG gamma chain. The VL DNA consisted of 312 bases, coming from the mouse IgG kappa chain. The VH and VL genes were spliced by 45 bases coding a (G4S)3 flexible linker. The ScFv gene consisted of 696 bases. The ScFv antibody expressed by BL-21 (DE3) fused with TrxA tag protein and formed inclusions. The relative molecular mass of TrxA-ScFv fusion protein is about 40 x 10(3) and that of ScFv is about 24 x 10(3). The ScFv antibody has excellent activity tested by competitive inhibition ELISA, the TrxA-ScFv could inhibit about 41% of the McAb to bind antigen and ScFv could inhibit about 53%. CONCLUSION: We have successfully constructed an anti-human AFP ScFv gene with 696 bases; it can express in BL-21 with high activity.

Antibodies, Monoclonal↗
PubMed2004

Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris.

Candida rugosa lipase, a significant catalyst, had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications. Several isozymes encoded by the lip gene family, namely lip1 to lip7, possess distinct thermal stability and substrate specificity, among which the recombinant LIP1 showed a distinguished catalytic characterization. In this study, we utilized PCR to remove an unnecessary linker of pGAPZalphaC vector and used overlap extension PCR-based multiple site-directed mutagenesis to convert the 19 non-universal CTG-serine codons into universal TCT-serine codons and successfully express a highly active recombinant C. rugosa LIP1 in the Pichia expression system. Response surface methodology and 4-factor-5-level central composite rotatable design were adopted to evaluate the effects of growth parameters, such as temperature (21.6-38.4 degrees C), glucose concentration (0.3-3.7%), yeast extract (0.16-1.84%), and pH (5.3-8.7) on the lipolytic activity of LIP1 and biomass of P. pastoris. Based on ridge max analysis, the optimum LIP1 production conditions were temperature, 24.1 degrees C; glucose concentration, 2.6%; yeast extract, 1.4%; and pH 7.6. The predicted value of lipolytic activity was 246.9+/-39.7 U/ml, and the actual value was 253.3+/-18.8 U/ml. The lipolytic activity of the recombinant LIP1 resulting from the present work is twofold higher than that achieved by a methanol induction system.

Base Sequence↗
PubMed1998

Molecular analysis of Physarum haemagglutinin I: lack of a signal sequence, sulphur amino acids and post-translational modifications.

The cDNAs encoding haemagglutinin I from plasmodia of Physarum polycephalum have been cloned using PCR protocols. The composite haemagglutinin I cDNA sequence, derived from several overlapping clones from PCR fragments, spans 408 nt and the 315 bp ORF encodes a polypeptide of 104 aa without a typical signal sequence. The putative molecular mass deduced from the amino acid sequence (10,760.76 Da) corresponds exactly to that determined by electrospray ionization MS (10,759.86 +/- 0.15 Da), suggesting that haemagglutinin I is not subject to post-translational modification. Haemagglutinin I lacks sulphur amino acids and has a beta-sheet as the major secondary structure. Expression of the coding sequence in Escherichia coli yielded a product that exhibits the same sugar-binding specificity as natural haemagglutinin I. The deduced amino acid sequence shows little similarity to that of any known lectins and thus apparently represents a novel type of lectin.

Amino Acid Sequence↗
PubMed2003

[CYP21 gene point mutations study in 21-hydroxylase deficiency patients].

OBJECTIVE: The major cause of congenital adrenal hyperplasia (CAH) is 21-hydroxylase deficiency, which accounts for 90% - 95% of all cases in most populations. This study was conducted to characterize the molecular basis of the 21-hydroxylase deficiency and to obtain the spectrum of the CYP21 gene mutations in a group of Chinese patients, and analyze the relationship of genotype and phenotype. METHODS: To detect the distribution of gene mutations in Chinese population samples from 52 patients with 21-hydroxylase deficiency from 51 families were collected, including two parents samples in 30 patients and one parents sample in 10 patients. Blood samples were obtained for extraction of peripheral blood lymphocytes. Polymerase chain reaction (PCR) followed by nesed PCR were used to study the 21-hydroxylase gene (CYP21) mutations. The primary PCR amplified two overlapping CYP21-specific DNA fragments, The product of the nested PCR which used products from the primary PCR was analysed by restriction fragment length polymorphism (RFLP) or amplification-created restriction site (ACRS). All patients were studied by 6 mutations, including P30L, I2g (intron 2 nt 656 c/a-->g splice mutation), E3Delta8nt (exon 3 codon111-codon113 8 bp deletion), I172N, V281L and Q318X. RESULTS: Through analysis of 52 patients with 21-hydroxylase deficiency, in 5 patients no mutations were detected, in 17 patients only one mutated allele could be characterized, two different mutations were identified in 21 patients, three mutations were detected in 2 patients. Totally, in 73% of alleles the genotypes could be detected. The most common mutation was I2g, which present on 31% affected alleles, then followed by I172N, Q318X, V281L, P30L, E3Delta8nt, accounting for 23%, 14%, 9%, 3%, 2% of all identified mutations respectively, which included multiple mutations accounting for 6%. The most frequent molecular defects of the salt-wasting form were the I2g (45.7%), Q318X (26%). Of the simple virilizing form, the dominant mutations were I172N (40.7%) and I2g (18.5%). CONCLUSION: Six different mutations were examined in this study, and the detected mutations accounted for 73% affected alleles, in which I2g and I172N were the most common mutations (accounting for 54%). Correlation between genotypes and phenotypes was compatible with the reported data. Two rounds of PCR followed by RFLP or ACRS analysis may provide important information for genetic counseling and for prenatal diagnosis.

Adrenal Hyperplasia, Congenital↗
PubMed2006

Construction of an infectious cDNA clone of Ribgrass mosaic virus Shanghai isolate and its modification to express an epitope of Mycobacterium tuberculosis.

Infectious cDNA clones of the Shanghai isolate of Ribgrass mosaic virus (RMV) were produced by joining four overlapping cDNA fragments and also in a single step by long template PCR. After inoculation of Nicotiana glutinosa with either RNA transcripts or the cDNA under the control of the CaMV 35S promoter, plants developed typical symptoms, and viral coat protein could be detected in them by Western blot analysis. However, compared to plants inoculated with purified viral RNA, lesions were fewer and appeared more slowly. An epitope of the Mycobacterium tuberculosis 31-kDa protein was inserted at the C-terminus of the viral coat protein by PCR using two overlapping fragments. The modified clone was also infectious and the foreign epitope could be detected serologically in the electron microscope and by Western blot analysis. The results demonstrate the potential of RMV as a viral gene vector.

Antigens, Bacterial↗
PubMed2005

[Prokaryotic expression and characterization of an anti-KDR single chain antibody].

AIM: To construct and express a single chain antibody (scFv) against human vascular endothelial growth factor (VEGF) receptor KDR and characterize its biological activity. METHODS: The restriction enzyme sites were added to the previously cloned V(H) and V(L) genes of mAb Ycom1D3 against KDR by PCR. The anti-KDR scFv gene was constructed by the splicing overlap extensive (SOE) PCR and then inserted into fusion expression vector pAYZH. The recombinant protein was expressed in E.coli 16C9 and purified with His-tag affinity chromatography. The specificity of the purified scFv was examined by ELISA and FACS. RESULTS: DNA sequencing indicated that the cloned scFv gene consisted of 729 bp, encoding 243 amino acids. After induction in low-phosphate medium of AP5, a new protein band with relative molecular mass (M(r)) of 30 000 appeared on gel of SDS-PAGE and on nitrocellulose membrane of Western blot, which was consistent with the theoretically predicted value. Anti-KDR scFv was expressed in the form of inclusion body, which accounted for 20% of total bacterial protein. ELISA and competitive immunofluorescence binding test showed that the anti-KDR scFv had the same binding activity as mAb Ycom1D3 and that it could block VEGF/KDR interaction effectively. CONCLUSION: Recombinant anti-KDR scFv gene has been successfully constructed and expressed in E.coli 16C9, which lays the foundation for its diagnostic and therapeutic application.

Animals↗
PubMed2005

Molecular haplotyping by linking emulsion PCR: analysis of paraoxonase 1 haplotypes and phenotypes.

Linking emulsion PCR (LE-PCR) enables formation of minichromosomes preserving phase information of two polymorphic loci, hence the haplotype. Emulsion PCR confines two amplicons of two linked polymorphic sites on a single template molecule to one aqueous-phase droplet. Linking PCR uses biotinylated, overlapping linking primers to connect these amplicons in the droplet. After LE-PCR, unlinked amplicons are removed on streptavidin-coated magnetic beads and single-stranded runoff products are capped by primer extension. Quantitative ASPCR can then be used to ascertain the haplotypes of the two polymorphic loci on the minichromosomes. Using LE-PCR, we determined the human paraoxonase-1 [PON1] molecular haplotypes at three loci (-909g>c, L55M, Q192R) in women who were compound heterozygotes for -909g>c/L55M (n = 89), -909g>c/Q192R (n = 77) and L55M/Q192R (n = 68). We observed a strong association between PON1 substrate specificity (paraoxon/phenylacetate substrate activity ratios) and -909g>c/Q192R haplotype. We have demonstrated here a powerful molecular haplotyping technology that can be applied in population studies.

Aryldialkylphosphatase↗
PubMed1991

Seven novel Tay-Sachs mutations detected by chemical mismatch cleavage of PCR-amplified cDNA fragments.

Total RNA was isolated from cultured fibroblasts from 12 unrelated patients with Tay-Sachs disease, an autosomal recessive disorder due to beta-hexosaminidase A deficiency. beta-Hexosaminidase mRNA was amplified by cDNA-PCR in four overlapping segments spanning the entire coding sequence. In two patients, abnormal size cDNA-PCR fragments in which exons were removed resulted from splicing mutations that were characterized at the genomic DNA level: both were G to A transitions, at the first position of intron 2 and at the fifth position of intron 4. Five other mutations have been identified by cDNA-PCR chemical mismatch analysis and direct sequencing of an amplified fragment containing the mismatch site. One missense mutation alters the codon for Ser210 to Phe in exon 6 and the other one alters the codon for Arg504 to Cys in exon 13. A 3-bp deletion results in the deletion of a phenylalanine residue in exon 8. Two nonsense mutations in exon 3 (Arg137 to stop) and in exon 11 (Arg393 to stop) are associated with a marked decrease of mRNA abundance, probably because they result in mRNA instability. Three of the six single base mutations involve the conversion of a CpG dinucleotide in the sense strand to TpG. These results demonstrate the extreme molecular heterogeneity of mutations causing Tay-Sachs disease. The procedure described in this paper allows the rapid detection of any type of mutation, except those impairing the promoter function. Applicable even to patients with splicing or nonsense mutations and very low mRNA abundance, it has therefore a potentially broad application in human genetics, for both diagnostic and fundamental purposes.

Base Sequence↗