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Results for “Overlapping PCR”

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PubMed2003

Cloning and sequencing full-length HLA-B and -C genes.

Currently most available HLA-A, -B and -C DNA sequences cover exons 2 and 3 with a limited number extending to include other exons and introns. We have developed a method for the accurate determination of full-length genomic DNA sequences for HLA-A, -B and -C alleles. The method involves cloning of PCR amplified full-length HLA genes to separate alleles at heterozygous loci. The approach avoids any ambiguities from sequencing heterozygous PCR products directly and also avoids ambiguities from sequencing overlapping PCR products to achieve full-length sequence. To date we have sequenced full-length genomic sequences from representatives of all the major HLA-B and -C allele groups.

Alleles↗

PubMed1995

cDNA and deduced protein sequence of CYP6D1: the putative gene for a cytochrome P450 responsible for pyrethroid resistance in house fly.

A microsomal cytochrome P450 from the house fly (Musca domestica), termed P450lpr, is involved in P450 monooxygenase-mediated pyrethroid resistance and is expressed at 8-fold higher levels in the insecticide resistant LPR strain compared to a susceptible strain. An internal cDNA sequence was amplified by polymerase chain reaction (PCR) using degenerate primers based on known P450lpr polypeptide sequences, and the remainder of the sequence was amplified by single side-specific PCR. A 1.8 kb cDNA sequence was obtained from 3 overlapping PCR products, with an open reading frame encoding a P450 protein of 516 residues (M(r) 59,182). This gene has been designated CYP6D1 within the P450 gene superfamily. CYP6D1 exhibits most similarity (28.2-29.8% positional identity) to butterfly CYP6B1, house fly CYP6A1 and Drosophila CYP6A2, and also exhibits comparable similarity (24.7% identity) to rat CYP3A1. The deduced protein sequence contains a hydrophobic N-terminal region and conserved sequences thought to be involved in heme-binding and electron donor-protein interactions. Comparison of CYP6D1 with its four most similar proteins (CYP6B1, CYP6A1, CYP6A2 and CYP3A1) reveals the presence of extensive stretches of residues in an alignment row in 2 possible substrate-binding regions. Three introns of 74, 66 and 64 bp, having 5'-GT and AG-3' ends, split the CYP6D1 coding region in genomic DNA. Results indicate that CYP6D1 is likely the P450lpr gene.

Amino Acid Sequence↗

PubMed1991

Isolation and detection of urease genes in Ureaplasma urealyticum.

Urease from ureaplasmas was purified by immunoaffinity chromatography, and the N-terminal amino acid sequence was determined for two of the three subunits. These sequences were used to design primers for a polymerase chain reaction (PCR) that amplified most of the gene coding for one of the subunits. By using a novel "PCR walking" technique, we synthesized almost the complete locus on two overlapping PCR products. We present here a partial nucleotide sequence of the urease locus from Ureaplasma urealyticum (serotype 8), which agrees with our N-terminal amino acid data but differs slightly from the sequence previously reported (A. Blanchard, Mol. Microbiol. 4:669-676, 1990). Also described are PCR primers, intended for diagnostic use, that amplify a sequence from all Ureaplasma strains tested but not from any other mycoplasmas or urease-positive bacteria.

Amino Acid Sequence↗

PubMed2004

Cloning and characterization of the bovine testicular PH-20 hyaluronidase core domain.

The core nucleotide sequence of bovine (Bos taurus) testicular PH-20 hyaluronidase was cloned using one step RT-PCR. The 5' and 3' regions were cloned separately and a sequence overlap of 124 bp facilitated the fusion of these two fragments by overlapping PCR, resulting in a concatenated sequence of 1422 bp. This nucleotide sequence and its deduced amino acid sequence were compared to homologous sequences from eight other mammal species. The bovine sequences were most similar to those of the pig, Sus scrofa (swine Spam1: 79.1% nucleotide and 70.1% amino acid similarity) and least similar to sequences from the Norway rat, Rattus norvegicus (murine Spam1: 61% nucleotide and 53.3% amino acid similarity). A phylogenetic analysis joined the red fox (Vulpes vulpes) sequence as sister to the bull-pig pair. Twelve cysteine residues were conserved among all nine aligned amino acid sequences and five proposed glycosylation sites have been identified. The feasibility of developing an effective, low-cost bovine PH-20 expression system is discussed in light of these new data.

Amino Acid Sequence↗

PubMed1999

Construction of larger-size sequencing templates from degraded DNA.

We describe a modified recombinant PCR procedure that allows the construction of longer fragments of DNA from shorter, overlapping PCR products. This method permits the construction of optimal-size templates for DNA sequence analysis (500-600 bp) from DNA that is too degraded for direct amplification of fragments of this size. As an example, the entire hypervariable region 1 of human mtDNA (418 bp) was reconstructed in one step from five overlapping amplification products (159, 126, 131, 131 and 93 bp) derived from a Paleolithic tooth specimen.

Base Sequence↗

PubMed1997

Mutational analysis of the BRCA1 tumor suppressor gene in endometrial carcinoma.

OBJECTIVE: To investigate whether alteration of BRCA1 tumor suppressor gene occurs in sporadic endometrial carcinomas. METHODS: Genomic DNAs were prepared from 33 consecutively collected endometrial carcinoma tissues for BRCA1 mutational analysis. To screen for BRCA1 mutation, polymerase chain reaction (PCR) amplification and single strand conformation polymorphism (SSCP) technique were used with 41 overlapping PCR primer pairs for the 23 coding exons of BRCA1. Tumors that demonstrated SSCP variants were further subjected to direct DNA sequencing in the appropriate exons to identify the DNA alteration. RESULTS: In addition to detecting a previously described polymorphism in exon 11, single strand conformation polymorphism analysis of the 33 endometrial cancers identified 3 tumors with mobility shifts. Two tumors shifted in exon 3 and showed the same pattern of band shift. The other tumor shifted in exon 9. DNA sequencing revealed sequence alterations in the 3 tumors; all appeared heterozygous. In the 2 tumors shifted in exon 3, the sequence alteration caused no amino acid change and was consistent with an infrequent silent polymorphism. In the third tumor, a missense alteration at codon 191 was detected and was recognized as germline in origin. CONCLUSIONS: Because a normal allele of BRCA1 was retained in the tumor where a germline missense alteration was detected, the heterozygous DNA alteration should not be cancer predisposing in terms of the two-hit model for inactivation of the tumor suppressor gene. We conclude that mutation of BRCA1 may not be involved in the development of sporadic endometrial cancer.

DNA Mutational Analysis↗

PubMed2002

Brief communication: molecular analysis of the Kwäday Dän Ts'finchi ancient remains found in a glacier in Canada.

DNA was extracted from the frozen remains of a man found in the northwest corner of British Columbia, Canada, in 1999. His clothing was radiocarbon-dated at ca. 550 years old. Nitrogen and carbon content in whole bone and collagen-type residue extracted from both bone and muscle indicated good preservation of proteinaceous macromolecules. Restriction enzyme analysis of mitochondrial DNA (mtDNA) determined that the remains belong to haplogroup A, one of the four major Native American mtDNA haplogroups. Data obtained by PCR direct sequencing of the mtDNA control region, and by sequencing the clones from overlapping PCR products, were duplicated by an independent laboratory. Comparison of these mtDNA sequences with those of North American, Central American, South American, East Siberian, Greenlandic, and Northeast Asian populations indicates that the remains share an mtDNA type with North American, Central American, and South American populations.

Asian People↗

PubMed2006

Class 1 and class 2 integrons in non-prevalent serovars of Salmonella enterica: structure and association with transposons and plasmids.

OBJECTIVES: To characterize class 1 and class 2 integrons which were simultaneously detected in non-typhoid Salmonella enterica strains of non-prevalent serovars, and to investigate their possible association with transposons and/or plasmids. METHODS: Eight multidrug-resistant S. enterica strains belonging to serovars Virchow (4), Panama (2), Grumpensis (1) and Worthington (1), each containing a class 1 and a class 2 integron, were analysed. Nested PCR amplification was used to determine the gene-cassette configuration of the integrons. Overlapping PCR amplifications were applied in integron-transposon linkage experiments. Conjugation and hybridization experiments were used to localize integrons and transposons in the bacterial genome (plasmid and chromosome associated). RESULTS: One of two different class 1 integrons (with variable regions of 1000 bp/aadA1 and 2300 bp/sat-smr-aadA1) inserted into Tn21-like transposons, were found to coexist with the class 2 integron (2300 bp/dfrA1-sat1-aadA1) of Tn7 in the analysed strains. Class 1 integrons were always found in large conjugative plasmids whereas apparently intact or defective copies of the Tn7 integron could be located on the same plasmid and/or the bacterial chromosome. CONCLUSIONS: This report describes different associations between mobile genetic elements that play a crucial role in the capture and spread of antimicrobial drug resistance. As far as we are aware, this is the first description of class 2 integrons in serovars Panama, Grumpensis and Worthington.

Chromosomes, Bacterial↗

PubMed2003

GJB2 gene mutations causing familial hereditary deafness in Turkey.

Mutations in Connexin 26 (Cx26) play an important role in autosomal non-syndromic hereditary hearing loss. In this study, our objective was to find out the significance of Cx26 mutations in Turkish families who had hereditary deafness. Fourteen families who had at least two prelingually deaf children per family were included in the study. One affected child from each of the 14 families was selected for single-stranded conformational polymorphism SSCP analysis. Three PCR reactions were used for each subject to amplify the entire Cx26 coding region with overlap. PCR products were sequenced on an Applied Biosystems (ABI) model 3700 automated sequencer. Six of the 14 representative family members (42.9%) demonstrated shifts on SSCP and were subsequently sequenced for Exons 1 and 2 of GJB2 and were tested for the 432 kb upstream deletion. No mutations were found in Exon 1 and no 432 kb deletions were noted. Three different GJB2 mutations were found in Exon 2 of the probands, which were 35delG, 299-300delAT, and 487G > A (M163V). GJB2 mutations were detected in 21.4% of the families. Two patients were homozygous for 35delG and 299-300delAT mutations, and were given a diagnosis of DFNB1 deafness (14.3%). Two different polymorphisms, 457G > A (V153I) and 380G > AG (R127H) were also found. In conclusion, although GJB2 mutations were detected in 21.4% of the families tested, only 14.3% of subject representatives were homozygous and therefore deafness caused by Cx26 mutation segregated with DFNB1. Thus, contribution of GJB2 mutations appears less significant in familial deafness. This necessitates further assessment for the other known gene regions as well as a search for new genetic factors in familial type of genetic deafness.

Child↗

PubMed1994

Screening of genomic DNA to identify mutations in the gene for Bruton's tyrosine kinase.

Mutations in the gene for Bruton's tyrosine kinase (Btk) are responsible for X-linked agammaglobulinemia (XLA). Thus far, mutations in this gene have been identified based on alterations in Southern or Northern blot analysis or cDNA sequence. To permit detection of mutations in genomic DNA, we designed PCR primers to flank each of the 19 exons of Btk with splice sites. Two overlapping PCR products were employed for exons longer than 230 base pairs. Single strand conformation polymorphism (SSCP) analysis was used to screen PCR products from 30 unrelated families presumed to carry a Btk mutation. It was possible to amplify DNA in every reaction from every patient, indicating that large deletions in Btk are uncommon. Twenty three different mutations were found in 25 unrelated families, including one family in whom DNA was available from a carrier but not an affected patient. Seven mutations were single base pair substitutions resulting in premature stop codons scattered throughout the gene. Small insertions or deletions causing frameshifts and secondary premature stop codons constituted an additional seven mutations. One patient had a point mutation in the start codon and one patient had a mutation in a splice donor site. Point mutations resulting in amino acid substitutions were seen in nine patients. Northern blot analysis of RNA from three patients with premature stop codons showed an absence of Btk transcript whereas four patients with amino acid substitutions had normal amounts of transcript of normal size. These studies document the considerable variability in the Btk mutations causing XLA and they demonstrate an approach that will be useful for carrier detection as well as mutation identification.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed2003

[Analysis of suppressive roles of BLU gene at 3p21.3 in nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: Nonrandom allelic loss at chromosome 3p21.3 is a common and early event in nasopharyngeal carcinoma (NPC), which implicates the presence of tumor suppressor genes (TSGs) that may be involved in the pathogenesis of NPCs. BLU gene, containing a MYND domain and located at 3p21.3, has been considered as a NPC associated candidate tumor suppressor gene (TSG) due to the occurrence of loss of its expression and aberrant promoter hypermethylation in most NPCs. This study was designed to construct expression vectors containing either wild type BLU gene and its mutants and to analyze the effect of BLU gene on proliferation of NPC cells by transfection assays. METHODS: The full-length cDNA of BLU gene was amplified by RT-PCR. The expression vectors containing various BLU mutants were constructed by site-directed mutagenesis by overlapping PCR. These mutants include a MYND domain deletion mutant, a Ser402Phe and del405Cys, del406Ser mutant, and a Gly160Arg mutant. The wild type BLU gene and the MYND domain deletion mutant were transfected into NPC cell lines CNE1 and CNE2. The effect on apoptosis was determined by TUNEL assay. Cellular proliferation of the stably-transfected cells was examined with cell growth curve and by colony formation assays. Tumorigenicity in nude mice of CNE2 stably-transfected with BLU was investigated. RESULTS: No significant difference in apoptosis index (AI) was observed between cells transfected with wild type or MYND domain deleted BLU gene and cells transfected with plasmid controls. Exogenous expression of wild type BLU gene had no effect on growth rate and colony formation ability of CNE1 and CNE2. BLU gene showed no suppressor ability in CNE2 tumorigenicity. CONCLUSION: Although BLU gene was frequently altered in NPCs, its suppressor role in NPC cells proliferation was not evident. Thus, the possibility of BLU gene as a TSG involved in NPC development remained to be elucidated by further studies.

Animals↗

PubMed2002

Revisiting the yeast chromosome VI DNA sequence reveals a correction merging YFL007w and YFL006w to a single ORF.

The overall contiguity of the Saccharomyces cerevisiae chromosome VI sequence assembly was assessed by systematic long-range PCR, PCR product size determination and sequencing. Using S. cerevisiae strain FY1679 total genomic DNA as template, 41 overlapping PCR products were generated, covering the complete 270 kb chromosome VI sequence. Specificity of the PCR products was confirmed by direct end-sequencing. No fragment size discrepancies with the published sequence were observed, confirming the overall sequence assembly. Gel reads of the PCR-fragment ends compile to a total of resequenced DNA representing 16% of the entire chromosome VI and reveal three single nucleotide differences. One of these is an extra G in the protein-coding region of YFL007w. Due to this additional nucleotide, the coding sequences of YFL007w and YFL006w become part of a 6432 bp ORF. The same sequence also resulted from analysis of strain BY4743. Homologous proteins of unknown function found in Candida albicans, Arabidopsis thaliana, Caenorhabditis elegans and man, as well as comparative data from published transcript profiles of YFL006w and YFL007w, give additional evidence for the existence of a single gene at this locus in yeast.

Amino Acid Sequence↗

PubMed2006

[Construction and preliminary application of a human natural phage antibody library with diversity].

AIM: To construct a human natural phage single-chain antibody (scFv) library with diversity. METHODS: V(H) and V(L) genes were amplified by RT-PCR and hemi-PCR from peripheral blood lymphocytes of healthy persons. The V genes were assembled to form scFv by overlap PCR and cloned into phagemid pCANTAB-5E, and then transformed into E. coli TG1 by electroporation to construct a human natural phage scFv library. The diversity and gene family of antibody gene were analysed by sequencing and the specific antibodies against various antigens were screened through bio-panning. RESULTS: A human natural phage scFv library with diversity and 2x10(8) sink size was constructed successfully. The specific human scFvs against 5 antigens were obtained by bio-panning. CONCLUSION: A human natural phage scFv library with diversity is constructed successfully and can be applied to human antibody preparation.

Antibodies↗

PubMed1996

Novel mutations detected in the TSC2 gene from both sporadic and familial TSC patients.

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by hamartomas in one or more organs, including the brain, skin, heart and kidneys. Linkage studies have shown locus heterogeneity with one TSC gene mapped to chromosome 9q34 and a second to 16p13.3. The gene on 16p13.3, TSC2, has been cloned and shown to encode a 5.5 kb transcript that is widely expressed. To facilitate the search for mutations in the TSC2 gene product, tuberin, we have designed an RT-PCR-based assay system to scan the expressed coding region of the TSC2 gene in lymphoblasts. Using 34 overlapping PCR assays we performed single-strand conformation polymorphism analysis of DNA from 26 apparently sporadic TSC cases, two TSC families non-informative for linkage analysis and two confirmed chromosome 16-linked TSC families. Of the 60 chromosomes scanned, 14 showed abnormal SSCP mobility shifts. Using direct PCR sequencing we have identified five missense mutations, one 3 bp in-frame deletion and one 2 bp frameshift deletion, one nonsense mutation, one 29 bp tandem duplication and five silent nucleotide changes that are likely to be polymorphisms. There is no apparent clustering of mutations within TSC2. The diversity of mutation types argues that TSC2 may not act in a classic tumor suppressor fashion. In addition, we saw no specific correlation between the different mutations and clinical severity or expression. These data confirm that TSC2 is indeed the relevant gene, and that a substantial number of sporadic cases arise from mutations in the TSC2 gene.

Base Sequence↗

PubMed2000

The ribosomal DNA of the Zygomycete Mucor miehei.

The ribosomal DNA-from the Zygomycete Mucor miehei has been characterised. The complete rDNA unit was cloned by heterologous PCR using primers whose sequence matched conserved regions of the rDNA from related fungal species. The sequence of the overlapping PCR products revealed the existence of a repeated unit of 9574 bp. The genes encoding the different rRNA species were identified by their homology to the corresponding sequences from other fungi. We estimate that the rDNA unit is present in the genome of M. miehei in about 100 copies. This estimation was made by comparing the intensity of its hybridisation signal in a Southern blot with that of the mmp gene coding for aspartyl protease, which was assumed to be contained in single copy. The size and structure of the M. miehei rDNA unit was similar to that of other fungi. The genes encoding the 25S, 18S and 5.8S RNAs are closely linked within the repeated unit which also contains the 5S gene. This latter gene appears to be transcribed in the opposite direction. The 25S, 18S and 5.8S genes showed 70-80% homology to the corresponding genes from other fungi, whereas the degree of homology for the 5S gene was much lower. The highest homology (about 80%) corresponded to the few available sequences from other Mucor species. Homology to genes from other Zygomycota was no higher than that observed for genes from the Ascomycota or Basidiomycota fungi.

Base Sequence↗

PubMed1993

PCR mediated detection of a new human receptor-tyrosine-kinase, HEK 2.

We have previously amplified cDNA subfragments of protein-tyrosine-kinases (PTKs) by using the polymerase chain reaction (PCR) and specific sets of oligonucleotide primers derived from nucleotide sequences of their kinase domain. In this study we have used a more directed approach to identify new members of the EPH/elk-family by PCR of human embryonic cDNA: we utilized oligonucleotide primers specifically designed to a highly conserved N-terminal motif and the kinase region of EPH/elk-PTKs in RNA-PCRs. The 5' and 3' elongation of the primary PCR product was achieved by the RACE (rapid amplification of cDNA ends)-technique. Sequence analysis of 3.8 kb of overlapping PCR products allowed to identify a novel receptor-PTK, HEK 2 (human embryo kinase 2), as an additional member of this family, without the need to screen a cDNA library. This approach should be useful for the rapid isolation of other PTK-genes as well. Analysis of genomic DNA placed HEK 2 on chromosome 3. Northern blot analysis demonstrated the expression of a 4.6 kb HEK 2-mRNA in lung, brain, pancreas, liver, placenta, kidney, skeletal muscle, heart and several human cells. In a protein kinase assay with HEK 2-specific immunoprecipitates from the human epidermoid carcinoma cell line A431, a protein of 130 kDa was found phosphorylated.

Amino Acid Sequence↗

PubMed1994

Structure of mouse protein S as determined by PCR amplification and DNA sequencing of cDNA.

The cDNA sequence of mouse protein S was derived by conventional PCR amplification from liver mRNA, initially using primers derived from the human cDNA sequence, followed by direct DNA sequencing. Seven overlapping PCR fragments covering all of the mature protein, part of the propeptide, and the 3' noncoding region were generated and sequenced. In some cases primers based upon the human cDNA sequence were ineffective. Subsequent successful amplification with mouse-derived primers to the same regions and comparison of the mouse and human sequences in these regions suggest that the failure of the human primers was due to insufficient degree of heterospecies identity. The mouse protein S cDNA sequence of the coding region shares 82% identity to human. The 3' noncoding region of mouse protein S cDNA has several small deletions and insertions compared to human protein S cDNA. Mature mouse protein S consists of 634 amino acids in a single polypeptide chain and displays domain organization similar to that for other species. The amino acid sequence of mouse protein S is about 80% identical to that of other species. Eleven glutamic acid residues were found in the amino terminal region and are predicted to be sites of gamma-carboxylation. Amino acid residues #80-244 are defined as four cysteine-rich repeat sequences homologous to epidermal growth factor. The remainder of the molecule is homologous to plasma sex steroid binding protein. The mouse protein S contains two potential N-glycosylation sites at positions #458 and 468 and is lacking the putative glycosylation site at #490 found in human protein S.

Amino Acid Sequence↗

PubMedDate not supplied

A novel strategy for generation of monoclonal antibodies from single B cells using rt-PCR technique and in vitro expression.

Monoclonal antibodies (Mabs) are important biomolecules in immunology and have widespread applications in prognosis, diagnosis, and therapeutics. Here, we describe a novel approach called single-cell RT-PCR-linked in vitro expression (SICREX), which enables the high-throughput generation and screening of Mabs. This approach entails the isolation of B cells from immunized mouse spleen or human peripheral blood using magnetic microbeads conjugated with a B-cell-selective marker, anti-CD19. The light chain (Lc) and Fd portion of heavy-chain (Hc) genes of each cell are separately amplified by RT-PCR and then combined with the sequences of a T7 promoter, a ribosome binding site (rbs), and a T7 terminator by an overlapping PCR technique. The paired full-length DNA fragments of Lc and Hc genes from single B cells are simultaneously expressed by an Escherichia coli in vitro transcription and translation system followed by an enzyme-linked immunosorbent assay to find positive fragments possessing the affinity for the antigen. From spleen cells of an immunized mouse with calcium binding protein 40, a Fab fragment with K(d) of 1.6 (+/- 0.3) x 10(-8) against the antigen was obtained. From human peripheral blood, Fab fragments against a blood group B-BSA were obtained in a similar manner. The SICREX approach is simple, rapid and versatile, allowing the high-throughput generation of naturally paired Lc and Hc with antigen-binding activity from various animal sources.

Amino Acid Sequence↗