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Results for “Overlapping PCR”

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At least 523 records · Page 29Linked to original sources

PubMed1998

Isolation of two functional retinoid X receptor subtypes from the Ixodid tick, Amblyomma americanum (L.).

Retinoid X receptors (RXR) play a central role in a variety of nuclear signaling pathways in both vertebrates and invertebrates. Vertebrate RXRs are encoded by a multigene family whereas the insect RXR homologue, ultraspiracle (USP), is encoded by a single gene. To determine if acarines possess an RXR homologue similar to insect USPs, we isolated cDNAs encoding two distinct RXR genes, AamRXR1 and AamRXR2, from the ixodid tick, Amblyomma americanum (L.). The DNA binding domains share 95 and 87% identity, respectively, with DNA binding domains from insect USP and vertebrate RXR proteins. However, the ligand binding domains of the AamRXRs are more similar to vertebrate RXRs than to insect USP ligand binding domains (approximately 71 vs approximately 52%). Northern blot and RT-PCR analysis reveal both unique and overlapping patterns of AamRXR1 and AamRXR2 expression. Transactivation analysis show that both AamRXRs encode proteins which can form functional ecdysteroid receptors but are unlikely to bind retinoic acids.

Alternative Splicing↗

PubMed1997

The screening of 13 short tandem repeat loci in the Chinese population.

Population studies of 13 short tandem repeat (STR) loci were carried out on Chinese in Taiwan. The STR loci included HUMF13B, HUMF13A01, HUMFES/FPS, HUMFABP, HUMPLA2A1, HUMTPOX, HUMTH01, HUMVWFA31/A, HUMCSFIPO, HUMLPL, HUMGPP3A09, HUMCYAR04 and HUMCD4. DNA samples from 100 unrelated individuals were screened. The STR allele patterns were detected by the fluorescence detector of an automated DNA sequencer. Two PCR amplifications were performed for each STR locus in this study. The first PCR amplification strategy used 26 base pairs of the T7 sequence extension in the 5' end of the forward primer of each STR locus. The second PCR amplification used a dye-labeled T7 primer instead of the forward primer in the first PCR amplification, and the first PCR products as template to produce fluorescent dye-labeled PCR products. PCR products of different STR loci with overlapping allele sizes could be detected in the same lane of the polyacrylamide gel on an automated DNA sequencer using different colored dye-labeled T7 primers. There was no need to directly conjugate the fluorescent dye to individual STR primers. The PCR products were obtained using 2 ng of template DNA in 25 microliters of PCR reaction mixture. No deviations from the Hardy-Weinberg equilibrium were observed for the 13 STR loci. The distributions of these STR alleles were different from those of Caucasians or Blacks. The probability of matching from the combination of the 13 STR loci was 5.9 x 10(-10) for our Chinese population. However, HUMF13B, HUMLPL and HUMCD4 loci were not as highly polymorphic as observed in other populations.

Alleles↗

PubMed1997

Overexpression of a zebrafish homologue of the Drosophila neurogenic gene Delta perturbs differentiation of primary neurons and somite development.

We describe here the isolation and characterization of a zebrafish Delta homologue (delta D). A PCR fragment was used to obtain overlapping cDNA clones encoding a protein of 717 amino acids with all characteristic features of proteins of this family, a signal peptide, a transmembrane domain, and an extracellular region comprising the DSL domain and eight EGF-like repeats. The gene is transcribed in a complex pattern in the developing nervous system as well as in the hypoblast. Overexpression of this gene following mRNA injections leads to a reduction in the number of islet-I positive cells, which are assumed to be primary neurons, and to various defects in the adaxial mesoderm, as well as in the somites and myotomes. This suggests that delta D, and the Notch signalling pathway are involved in the differentiation of primary neurons within the neural plate, as well as in somite development.

Amino Acid Sequence↗

PubMed2001

Molecular analysis of vanA enterococci isolated from humans and animals in northeastern Italy.

A total of 53 vancomycin-resistant vanA-positive enterococci isolates from poultry farms (17 Enterococcus faecium; 8 Enterococcus durans) and from different hospitals (23 E. faecium; 5 Enterococcus faecalis) in northeastern Italy were compared on the basis of their antibiotic susceptibilities, their SmaI pulsed-field gel electrophoresis (PFGE) patterns, and the organization of their Tn1546-related elements. Ampicillin resistance was similar in both groups of isolates (52 and 60.7%, respectively), whereas human strains were more resistant to high-level gentamicin and streptomycin. A total of 52% of animal strains and 60% of human strains were resistant to tetracycline, and 56% and 46.4% to quinupristin/dalfopristin, respectively. In E. faecium and E. durans animal isolates, nine and six distinct PFGE patterns, respectively, were found: in two instances indistinguishable isolates were found from different farms. In E. faecium and E. faecalis human isolates, nine and six distinct PFGE patterns, respectively, were found; among E. faecium strains, 12 were identical or closely related and were isolates from the same hospital. Elements mediating vanA-glycopeptide resistance were characterized by PCR with primers that amplified 10 overlapping fragments of Tn1546. A total of 84.6% of animal strains and 64.2% of human strains contained elements indistinguishable from the prototype Tn1546. In addition, nine different types were identified, but none was common to animal and human strains.

Animals↗

PubMed1993

Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana.

A plant cytosine methyltransferase cDNA was isolated using degenerate oligonucleotides, based on homology between prokaryote and mouse methyltransferases, and PCR to amplify a short fragment of a methyltransferase gene. A fragment of the predicted size was amplified from genomic DNA from Arabidopsis thaliana. Overlapping cDNA clones, some with homology to the PCR amplified fragment, were identified and sequenced. The assembled nucleic acid sequence is 4720 bp and encodes a protein of 1534 amino acids which has significant homology to prokaryote and mammalian cytosine methyltransferases. Like mammalian methylases, this enzyme has a C terminal methyltransferase domain linked to a second larger domain. The Arabidopsis methylase has eight of the ten conserved sequence motifs found in prokaryote cytosine-5 methyltransferases and shows 50% homology to the murine enzyme in the methyltransferase domain. The amino terminal domain is only 24% homologous to the murine enzyme and lacks the zinc binding region that has been found in methyltransferases from both mouse and man. In contrast to mouse where a single methyltransferase gene has been identified, a small multigene family with homology to the region amplified in PCR has been identified in Arabidopsis thaliana.

Amino Acid Sequence↗

PubMed1998

Detection of new human papillomavirus sequences in skin lesions of a renal transplant recipient and characterization of one complete genome related to epidermodysplasia verruciformis-associated types.

Human papillomavirus (HPV) DNA, originally isolated from patients suffering from the skin disease epidermodysplasia verruciformis (EV), and a growing number of related sequences have recently been detected in a high percentage of benign and malignant skin lesions of both immunosuppressed and immunocompetent people. HPV L1 DNA fragments (374-389 bp long) from a solar keratosis and a squamous cell carcinoma (SCC) of a renal transplant recipient were amplified, cloned and sequenced. In 54 clones, six different HPV sequences were identified. One of these six corresponded to the known type HPV-8 and two (RTRX3 and RTRX7) have been described previously in cutaneous lesions of immunosuppressed patients. The remaining three sequences were different from all known HPV types: an HPV-9-related sequence (77.4% identity), an RTRX2-related sequence (82.6% identity), and an HPV-22-related sequence (83.7% identity). These three sequences, representing putatively new HPV types, were named RTRX8, RTRX9 and RTRX10, respectively. RTRX7 was found in the majority of clones from both lesions. The complete genome of RTRX7 (7731 bp) was cloned as six overlapping subgenomic fragments, generated by nested PCR with DNA extracts from the SCC. RTRX7 showed a genome organization typical of HPVs associated with EV. The L1 DNA sequence differed by 15% from the corresponding region of its closest known relative, HPV-12; thus, RTRX7 can be regarded as a new HPV type. RTRX7 DNA could not be detected by Southern blot hybridization with the homologous probe, indicating that the DNA concentration was below one copy per 10 cells in the investigated SCC.

Base Sequence↗

PubMed1993

Cloning and sequence analysis of the dnaK gene region of Lactococcus lactis subsp. lactis.

A 5.4 kb HindIII fragment of Lactococcus lactis subsp. lactis was identified using a homologous dnaK probe generated by PCR and cloned in Escherichia coli. Upstream sequences were generated by inverse PCR. The two cloned fragments partially overlapped, and sequencing of 5915 bp revealed the presence of four open reading frames in the order orf1-grpE-dnaK-orf4. orf1 encodes a 39 kDa protein of unknown function which shows considerable sequence homology with the Orf39 and Orfa proteins of Bacillus subtilis and Clostridium acetobutylicum, respectively. The downstream ORFs showed high homology to the grpE and dnaK genes of other prokaryotes. The DnaK protein has a characteristic 24-amino-acid deletion exhibited by all the known DnaK proteins of Gram-positive species. In many bacteria the dnaK and dnaJ genes are found as part of the same operon. The L. lactis dnaK operon is unusual in that the dnaK gene is followed by a putative transcription terminator and a fourth large ORF which shares no homology with the dnaJ genes of other bacteria but has a small degree of homology with various membrane proteins. Vegetative promoter sequences are found upstream of both orf1 and orf4. A 12 bp inverted repeat is found upstream of the putative promoter of orf1 and an 8 bp inverted repeat is found between this promoter and the orf1 initiation codon. These repeats are thought to be involved in regulation of the heat-shock genes. The DnaK homologue is induced approximately 3-fold on heat shock at 42 degrees C.

Amino Acid Sequence↗

PubMed1992

Direct sequencing and bidirectional allele specific polymerase chain reaction of the bovine beta-casein B variant.

We have used the polymerase chain reaction (PCR) to amplify exon VII of the bovine beta-casein gene. The mutations responsible for the B variant were identified by direct sequencing of the amplification products. A bidirectional allele-specific PCR method (BAS-PCR) has been developed using oligonucleotides overlapping the mutation site at their 3' ends. This new procedure allows a rapid and reliable discrimination between the B and non-B alleles of beta-casein.

Alleles↗

PubMed1995

Direct sequencing of SSP-PCR-amplified cDNA to identify new alleles in the DR52-associated DRB1 group: identification of DRB11115, DRB11117 and DRB1*1319.

Low and high resolution sequence specific oligonucleotide probe hybridization patterns were used to design an approach to direct sequencing of allele specific amplified cDNA. Several PCR amplifications were used to derive overlapping sequence fragments to define complete first domain sequences for a single allele. This method has been used to characterize three new DRB1 alleles in the DR52 family, DRB1*1115, DRB1*1117, and DRB1*1319. All three alleles carry polymorphisms previously observed in other DRB alleles and underscore the importance of utilizing a directed sequencing approach for obtaining unambiguous typing results in matching for bone marrow transplantation between unrelated donor and recipient.

Alleles↗

PubMed2006

Characterization of naturally occurring and Lamivudine-induced surface gene mutants of hepatitis B virus in patients with chronic hepatitis B in India.

BACKGROUND: Besides vaccine escape or immune escape hepatitis B virus (HBV) mutants, naturally occurring and drug-induced mutations have been reported in the surface gene (S-gene) of HBV. AIM: To investigate the frequency and profile of naturally occurring S-gene mutants and the influence of long-term lamivudine therapy in patients with chronic hepatitis B (CHB). MATERIALS AND METHODS: 57 patients with histologically proven CHB, on lamivudine 100 mg/day for more than 24 months, were included. Viral DNA was extracted at baseline and from on-therapy serum samples. The region encoding the complete major hydrophilic region (MHR) and flanking regions (nucleotides 425-840) of major S-gene that overlapped with the viral polymerase was PCR amplified and sequenced. End-of-therapy response (ETR) was assessed. RESULTS: Two (3.5%) patients had naturally occurring HBV mutants, sP127S and sS143L seen in the 'a' determinant of the S-gene. Following lamivudine therapy, 14 of 57 (24.5%) patients developed 16 types of S-gene mutations (sP120S, sA128V, sS143L, sW182St., sT189I, sV190A, sS193L, sI195M, sW196L, sW196St., sS207R, sI208T, sS210E, sF219S, sF220L and sC221G). Thirteen (81.2%) of these mutations emerged downstream to the MHR. Nine of 16 types of S-gene mutations observed with lamivudine therapy were also associated with the corresponding changes in the polymerase gene. Baseline viral DNA was significantly higher (2,093 vs. 336 pg/ml; p < 0.05) among patients developing S-gene mutants and the ETR in them was significantly lower [3 of 16 (18.8%) vs. 17 of 41 (41.5%); p < 0.05]. CONCLUSIONS: Naturally occurring S-gene mutations are uncommon and are restricted to the 'a' determinant region. Mutations develop in about a quarter of the patients on lamivudine therapy, mostly downstream to the MHR. They may contribute to non-response to the antiviral therapy.

Adult↗

PubMed2005

Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase.

AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase (AP). METHODS: The VH-linker-VL, namely scFv gene, was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by Sfi I and Not I, it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1. The positive colonies were screened by colony PCR and their expressions were induced by IPTG. ScFv gene was gained by digesting ScFv expression vector pUC19/119 with Sfi I and Not I restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1. The positive colonies were selected by bacterial colony PCR. The expression of fusion protein (scFv-AP) was induced by IPTG. Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku. Immunofluorescent assay (IFA) demonstrated its reactivity with TfR. The molecular weight of scFv-AP was 75 ku. Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP. It is promising for immunological experiments.

Alkaline Phosphatase↗

PubMed2001

Evidence for an antigen-driven humoral immune response in medullary ductal breast cancer.

A minority of breast cancers is characterized by lymphoplasmacytic infiltrates that have been correlated with improved patient survivals. The association of improved prognosis with plasmacytic infiltrates has been classically linked with the rare medullary carcinoma subtype but is also evident in the smaller infiltrated fraction of the more abundant nonmedullary (not otherwise specified) tumors. It is our hypothesis that these plasma cell (PC) infiltrates represent a host humoral response driven by one or more tumor-derived neoantigens. As the index study group, two primary medullary carcinoma tumors were examined. Immunophenotyping confirmed a large number of IgG PCs in contradistinction to normal breast, which typically contains a lesser number of mainly IgA isotypes. IgG heavy and light chains were expressed as combinatorial phage Fab libraries. VH and VL sequences showed a preponderance of clonal groups in both patients, as identified by germ-line gene usage and junctional mutation patterns. Panning of phage Fab libraries against purified antigens excluded Her2/neu and p53 as the eliciting antigen, and failure of clonal enrichment by cell panning suggested that the neoantigen was not membrane expressed or was expressed at low levels. Cognate, original VH+VL pairs were obtained by single cell PCR of tumor PCs, which showed overlap with the pooled IgG libraries. Tumor-derived IgG V genes exhibited mutational patterns that were consistent with antigenic selection and affinity maturation. Where examined, IgG1 was the predominant isotype, consistent with a T-dependent (i.e., protein) antigen. From these data, we infer that the breast tumor PC infiltrates of the medullary carcinoma subtype are compatible with an autogenic tumor neoantigen-driven humoral immune response.

Antibodies, Neoplasm↗

PubMed1998

Identification of a novel metastasis-suppressor region on human chromosome 12.

There is a critical need for markers that can be used to predict accurately the malignant potential of histological prostate cancers (J. T. Isaacs. Am. J. Pathol., 150: 1511-1521, 1997). Metastasis-suppressor genes are attractive candidates for marker development because, by definition, their loss should be associated with the acquisition of metastatic ability. In an effort to identify such genes, a single copy of human chromosome 12, tagged with the neomycin resistance gene, was introduced into highly metastatic Dunning AT6.1 prostate cancer cells by microcell-mediated chromosomal transfer. Thirty-two AT6.1-12 clonal cell lines were established and the region(s) of chromosome 12 retained was determined by sequence tagged site-based PCR analysis. Representative AT6.1-12 clones containing overlapping regions of chromosome 12 were characterized cytogenetically and were shown to have a normal complement of parental AT6.1 rat chromosomes. Fluorescence in situ hybridization, performed on representative AT6.1-12 hybrids, demonstrated a single human chromosome 12-specific signal. The metastatic ability of six representative clones was tested in immunodeficient mice. All of the AT6.1-12 clones showed the same in vivo growth rates as the control AT6.1-neo cells. Clonal cell lines that contained a conserved approximately 70-cM portion of chromosome 12 (e.g., AT6.1-12-8, -8-1, and -8-3), showed a >30-fold suppression in the number of macroscopic surface lung metastases. Mice that received injections of these cells developed a mean number 4 lung metastases whereas mice that received injections of other AT6.1-12 hybrids (lacking the approximately 70-cM region) or AT6.1-neo control cells, developed a mean number of 140 metastases. Interestingly, histological examination of the lungs of the mice that received injections of AT6.1-12-8 cells showed essentially no microscopic metastases. These findings suggest that a gene(s) encoded by the approximately 70-cM portion of human chromosome 12 suppresses an early step in the metastatic cascade.

Animals↗

PubMed1994

Rapid and efficient construction of yeast artificial chromosome contigs in the mouse genome with interspersed repetitive sequence PCR (IRS-PCR): generation of a 5-cM, > 5 megabase contig on mouse chromosome 1.

We have developed a new technique for the generation of YAC contigs in the mouse genome that is based on the ability to detect overlapping clones by hybridization of shared IRS-PCR products. As a demonstration of the technique, a 5-cM, > 5 megabase contig was developed on the distal half of mouse Chromosome (Chr) 1, spanning the region from Lamb2 to At3. The contig covers roughly 5% of the genetic distance of the chromosome and is comprised of more than 80 clones; 71 probes were assigned physical order to the chromosome, of which 59 were new markers generated in this study. Eight of the new probes were shown to be polymorphic between C3H/HeJ-gld and M. spretus. Three probes were mapped on a [(C3H/HeJ-gld x M. spretus) x C3H/HeJ-gld] interspecific backcross to integrate the physical map with a high-resolution genetic map of the region.

Animals↗

PubMed2002

A Blumeria graminisf.sp. hordei BAC library--contig building and microsynteny studies.

A bacterial artificial chromosome (BAC) library of Blumeria graminis f.sp. hordei, containing 12,000 clones with an average insert size of 41 kb, was constructed. The library represents about three genome equivalents and BAC-end sequencing showed a high content of repetitive sequences, making contig-building difficult. To identify overlapping clones, several strategies were used: colony hybridisation, PCR screening, fingerprinting techniques and the use of single-copy expressed sequence tags. The latter proved to be the most efficient method for identification of overlapping clones. Two contigs, at or close to avirulence loci, were constructed. Single nucleotide polymorphism (SNP) markers were developed from BAC-end sequences to link the contigs to the genetic maps. Two other BAC contigs were used to study microsynteny between B. graminis and two other ascomycetes, Neurospora crassa and Aspergillus fumigatus. The library provides an invaluable tool for the isolation of avirulence genes from B. graminis and for the study of gene synteny between this fungus and other fungi.

Ascomycota↗

PubMed1999

MART-1 is expressed less frequently on circulating melanoma cells in patients who develop distant compared with locoregional metastases.

PURPOSE: Polymerase chain reaction (PCR) with tyrosinase and with MART-1 permits detection of small numbers of circulating melanoma cells (CMCs) in patients who have undergone surgical resection of localized disease. In a previous study, we showed that PCR with MART-1 had sensitivity and specificity similar to those of PCR with tyrosinase in terms of detection of CMCs but that PCR with MART-1 seemed to identify a different but overlapping subgroup of patients. In the current study, we examined the utility and prognostic significance of PCR with tyrosinase and with MART-1. PATIENTS AND METHODS: We analyzed the prognostic significance of the patterns of expression of tyrosinase and MART-1 in 186 patients followed sequentially before and after surgical removal of American Joint Committee on Cancer stage I, II, or III melanoma. RESULTS: PCR with tyrosinase and with MART-1 in the first 3 months after surgery identified 68.5% of 73 patients who developed recurrence in the 2-year period after surgery. Approximately 35% of patients with positive tests remained disease-free at 2-year follow-up. We found that patients with disseminated recurrence had a significantly lower incidence of MART-1-positive CMCs (16%) than of tyrosinase-positive CMCs (63%). Patients with locoregional metastases had CMCs that expressed tyrosinase and MART-1 at similar rates. These differences in expression of the markers in patients with disseminated recurrence were also associated with a much lower disease-free survival, in those who had CMCs that were positive for tyrosinase but negative for MART-1. The reverse applied in those with locoregional disease. CONCLUSION: These findings suggest that PCR with MART-1 and with tyrosinase identifies subgroups of patients who develop disseminated or locally recurrent metastases. We hypothesize that immune responses against MART-1 may reduce the establishment of disseminated metastases.

Antigens, Neoplasm↗

PubMed1999

Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis.

Here, we describe a new approach for mutational scanning of PCR products through hybridization analysis between complementary oligonucleotides. Sets of overlapping probe oligonucleotides complementary to wild-type (WT) sequence are hybridized to microbead-immobilized PCR products under solution-like conditions. Mismatch-hybridization situations between a mutant sample and probe oligonucleotides result in higher remaining concentrations in solution of involved probe oligonucleotides. Post-hybridization supernatants are subsequently analyzed for their probe oligonucleotide compositions using surface plasmon resonance-based biosensor technology. Relative remaining probe oligonucleotide concentrations are monitored in real-time through hybridization analysis between probe oligonucleotides and their corresponding sensor-chip immobilized complementary counterparts. This allows for the construction of composition diagrams revealing the existence and approximate location of a mutation within an investigated sample DNA sequence. Applied on PCR products derived from clinical samples of microdissected tumor biopsies, single mutations in exons 6 and 7 of the human p53 tumor-suppressor gene were successfully detected and approximately localized.

Biosensing Techniques↗

PubMed2005

Differentiation of herpes simplex virus types 1 and 2 in clinical samples by a real-time taqman PCR assay.

While the clinical manifestations of HSV-1 and -2 overlap, the site of CNS infection, complications, response to antivirals, frequency of antiviral resistance, and reactivation rate on mucosal surfaces varies between HSV-1 and -2. Detection of HSV DNA by PCR has been shown to be the most sensitive method for detecting HSV in clinical samples. As such, we developed a PCR-based assay to accurately distinguish HSV-1 from HSV-2. Our initial studies indicated the assay using type specific primers was slightly less efficient for detecting HSV-1 and -2 DNA than the high throughput quantitative PCR assay we utilize that employs type common primers to gB. We subsequently evaluated the type specific assay on 3,131 specimens that had HSV DNA detected in the type common PCR assay. The typing results of these specimens were compared with the monoclonal antibody staining results of culture isolates collected from the same patients at the same time, and the HSV serologic status of the patient. The typing assay accurately identified both HSV-1 and -2 with a specificity of >99.5% and was significantly more sensitive than typing by culture and subsequent monoclonal antibody assays. Complete concordance was seen between the typing assay and HSV serologic status of the patient. Dual (HSV-1 and -2) infection in clinical samples was recognized in 2.6% of clinical samples using the new typing assay. This assay, when used in combination with the type common assay, can now accurately type almost all mucosal and visceral HSV isolates by molecular techniques.

Antibodies, Monoclonal↗