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Results for “Overlapping PCR”

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At least 415 records · Page 23Linked to original sources

PubMed2005

Role of sequence variations of the GnRH receptor and G protein-coupled receptor 54 gene in male idiopathic hypogonadotropic hypogonadism.

OBJECTIVE: To determine the frequency of mutations of the gonadotropin-releasing hormone receptor (GnRHR) and of the G protein-coupled receptor 54 (GPR54) genes in normosmic idiopathic hypogonadotropic hypogonadism (IHH). METHODS: In a retrospective study we analyzed the GnRHR and the GPR54 genes of 45 IHH patients and 50 controls. Genomic DNA was amplified by PCR to obtain partially overlapping amplicons encompassing the exon-intron boundaries of the GnRHR and GPR54 genes and analyzed by single-stranded conformation polymorphism gel electrophoresis and/or DNA sequencing. RESULTS: One heterozygous R262Q mutation of the GnRHR gene was identified in one patient with familial IHH. The silent single-nucleotide polymorphism (SNP) 453C > T occurred at the same frequency in patients and controls. One patient with sporadic IHH and consanguineous parents showed a novel homozygous sequence variation of the GPR54 gene (1001_1002insC) resulting in an open reading frame shift and elongation of 43 amino acids with an increased number of proline residues in the intracellular receptor domain. This patient had delayed puberty, low testosterone (3.4 nmol/l), and low-normal LH and FSH levels responsive to GnRH. Pulsatile GnRH administration normalized testosterone levels and induced spermatogenesis sufficiently to induce a pregnancy with assisted reproduction. Two common SNPs in exon 1 and exon 5 of the GPR54 gene showed similar frequency distribution and hormonal profiles in IHH and controls. CONCLUSIONS: Mutations of the GnRHR and of the GPR54 gene are rare in IHH and should be investigated especially in cases with autosomal recessive transmission. Common SNPs of the GnRHR and GPR54 genes do not play any role in IHH.

Adolescent↗

PubMed2000

Nasopharyngeal angiofibroma: an APC-gene-associated tumor?

Nasopharyngeal angiofibromas are extremely rare, locally invasive tumors of unknown cause exclusively occurring in male adolescents. Recently, 6 cases have been reported in patients with familial adenomatous polyposis coli (Gardners syndrome). Mutations or allelic loss of the adenomatous polyposis coli (APC) gene have therefore been implied in the pathogenesis of nasopharyngeal angiofibroma. The authors analyzed 11 cases of nasopharyngeal angiofibromas from 9 male patients for mutations in the mutation cluster region and allelic loss of the APC gene. Six were primary tumors; 2 first recurrences; and 1, primary tumor with 2 recurrences. Direct sequence analysis was performed on several overlapping polymerase chain reaction (PCR) products. Detection of allelic loss was performed by restriction length polymorphism analysis at a polymorphic locus. No mutation was detected in 11 tumors of 9 different patients. None of the 9 informative (heterozygous) cases carried an allelic loss. We conclude that alterations of the APC gene do not play a major role in the development of nasopharyngeal angiofibroma. The coincidence of nasopharyngeal angiofibromas and adenomatous polyposis coli in some families implies the possibility that another gene in this region might be responsible for the development of nasopharyngeal angiofibromas. HUM PATHOL 31:1411:1413.

Adolescent↗

PubMed1998

[Cloning and sequencing of cDNA in NS3 region of Chinese hepatitis G virus isolated from Henan Province].

To study the genetic characteristics of non-structural region 3 of HGV in China, a HGV-RNA positive sample was collected from Henan. HGV RNA was extracted and amplified by RT-nested-PCR. We got four overlap fragments and they were cloned into plasmid vector pcDNA II. The cDNA was sequenced by the Sanger's method. Nucleotide homology between Henan strain and other reported HGV isolates (PNF2161, R10291, GBV-C) was 85.7% to 93.8% over the 2137 nucleotides examined. Comparison of amino acid sequence deduced from Henan strain with that of other isolates showed 96.1% to 97.7% homology. Three amino acid variations were found in NS3 region of all the three Chinese isolates of HGV. In addition, a N-glycosylation site was found in the NS3 region of all the six isolates.

Amino Acid Sequence↗

PubMed1994

Primary sequence of an alternatively spliced form of CR1. Candidate for the 75,000 M(r) complement receptor expressed on chimpanzee erythrocytes.

Chimpanzee erythrocytes express a 75,000 M(r) complement receptor (E-CR) that binds C3b bearing immune complexes and is recognized by an anti-CR1 mAb (E11). Human erythrocytes express the type 1 CR (CR1), the most common form being 220,000 M(r) and consisting of 30 short consensus repeats (SCRs) for its entire extracellular region. The purpose of this investigation was to determine the structure of the 75,000 M(r) chimpanzee E-CR. A chimpanzee cell line was identified that expressed a 220,000 M(r) CR1, and a 75,000 M(r) molecule that was recognized by E11 and could bind human C3i. Utilizing this cell line, chimpanzee CR1 cDNA was amplified in overlapping segments by the PCR, using primer pairs specific for various regions of human CR1 cDNA. Direct sequencing of the PCR-amplified products revealed 6044 nucleotides encoding the entire 220,000 M(r) chimpanzee CR1. This nucleotide sequence was 98.8% homologous to that of the human 220,000 M(r) CR1. Amplification using a CR1 primer from the signal peptide and from the cytoplasmic region yielded a 1985-bp PCR product, termed CR1a. The CR1a sequence was identical with the sequence encoding SCRs 1 to 6, SCRs 28 to 30, and the transmembrane and cytoplasmic regions of chimpanzee CR1. This alternatively spliced product of chimpanzee CR1 would encode a protein of 71,000 peptide m.w. with six potential N-glycosylation sites. Amplification employing a CR1 primer from SCR 1 and from the 3' untranslated region yielded a second PCR product of 1731 bp. This sequence, termed CR1b, encoded eight SCRs, followed by a hydrophobic region that ended in a stop codon. The first six SCRs of CR1b were closer in homology to the first six SCRs of a human CR1-like genomic sequence (97.4%) than to those of the chimpanzee CR1 (94.8%). Taken together, these sequence data suggest that the 75,000 M(r) chimpanzee E-CR is encoded by CR1a, an alternative splice variant of chimpanzee CR1. The CR1b is presumably derived from an RNA species related to the CR1-like genomic sequence previously described only in humans.

Alternative Splicing↗

PubMed1993

Association of transforming growth-factor alpha gene polymorphisms with nonsyndromic cleft palate only (CPO).

Genetic analysis and tissue-specific expression studies support a role for transforming growth-factor alpha (TGFA) in craniofacial development. Previous studies have confirmed an association of alleles for TGFA with nonsyndromic cleft lip with or without cleft palate (CL/P) in humans. We carried out a retrospective association study to determine whether specific allelic variants of the TGFA gene are also associated with cleft palate only (CPO). The PCR products from 12 overlapping sets of primers to the TGFA cDNA were examined by using single-strand conformational polymorphism analysis. Four DNA polymorphic sites for TGFA were identified in the 3' untranslated region of the TGFA gene. These variants, as well as previously identified RFLPs for TGFA, were characterized in case and control populations for CPO by using chi 2 analysis. A significant association between alleles of TGFA and CPO was identified which further supports a role for this gene as one of the genetic determinants of craniofacial development. Sequence analysis of the variants disclosed a cluster of three variable sites within 30 bp of each other in the 3' untranslated region previously associated with an antisense transcript. These studies extend the role for TGFA in craniofacial morphogenesis and support an interrelated mechanism underlying nonsyndromic forms of CL/P.

Adolescent↗

PubMed1990

Constructing DNA by polymerase recombination.

Polymerase-mediated recombination based on DNA polymerase chain reactions (PCRs) has been used to carry out directed joining at a present point of two DNA fragments initially contained in a plasmid and a single-stranded synthetic DNA. The process includes copying of these fragments by PCR with generation of an overlapping homologous region. Such overlap of 12 base pairs in length was found to be sufficient to provide further DNA joining also by use of PCR.

Base Sequence↗

PubMed1999

Large-scale survey of Campylobacter species in human gastroenteritis by PCR and PCR-enzyme-linked immunosorbent assay.

A PCR-based study of the incidence of enteropathogenic campylobacter infection in humans was done on the basis of a detection and identification algorithm consisting of screening PCRs and species identification by PCR-enzyme-linked immunosorbent assay. This was applied to DNA extracted from 3,738 fecal samples from patients with sporadic cases of acute gastroenteritis, submitted by seven regional Public Health Laboratories in England and Wales over a 2-year period. The sending laboratories had cultured "Campylobacter spp." from 464 samples. The PCR methodologies detected 492 Campylobacter-positive samples, and the combination of culture and PCR yielded 543 Campylobacter-positive samples. There was identity (overlap) for 413 samples, but 79 PCR-positive samples were culture negative, and 51 culture-positive samples were PCR negative. While there was no statistically significant difference between PCR and culture in detection of C. jejuni-C. coli (PCR, 478 samples; culture, 461 samples), PCR provided unique data about mixed infections and non-C. jejuni and non- C. coli campylobacters. Mixed infections with C. jejuni and C. coli were found in 19 samples, and mixed infection with C. jejuni and C. upsaliensis was found in one sample; this was not apparent from culture. Eleven cases of gastroenteritis were attributed to C. upsaliensis by PCR, three cases were attributed to C. hyointestinalis, and one case was attributed to C. lari. This represents the highest incidence of C. hyointestinalis yet reported from human gastroenteritis, while the low incidence of C. lari suggests that it is less important in this context.

Campylobacter↗

PubMed1991

A single amino acid substitution in a common African allele of the CD4 molecule ablates binding of the monoclonal antibody, OKT4.

The CD4 molecule is a relatively non-polymorphic 55 kDa glycoprotein expressed on a subset of T lymphocytes. A common African allele of CD4 has been identified by non-reactivity with the monoclonal antibody, OKT4. The genetic basis for the OKT4- polymorphism of CD4 is unknown. In the present paper, the structure of the CD4 molecule from an homozygous CD4OKT4- individual was characterized at the molecular level. The size of the CD4OKT4- protein and mRNA were indistinguishable from those of the OKT4+ allele. The polymerase chain reaction (PCR) was used to map the structure of CD4OKT4- cDNAs by amplifying overlapping DNA segments and to obtain partial nucleotide sequence after asymmetric amplification. PCR was then used to clone CD4OKT4- cDNAs spanning the coding region of the entire, mature CD4 protein by amplification of two overlapping segments followed by PCR recombination. The nucleotide sequence of CD4OKT4- cDNA clones revealed a G----A transition at bp 867 encoding an arginine----tryptophan substitution at amino acid 240 relative to CD4OKT4+. Expression of a CD4OKT4- cDNA containing only this transition, confirmed that the arginine----tryptophan substitution at amino acid 240 ablates the binding of the mAb OKT4. A positively charged amino acid residue at this position is found in chimpanzee, rhesus macaque, mouse and rat CD4 suggesting that this mutation may confer unique functional properties to the CD4OKT4- protein.

Alleles↗

PubMed1997

Application of interspersed repetitive sequence polymerase chain reaction for construction of yeast artificial chromosome contigs.

Construction of physical maps across candidate regions is one of the rate-limiting steps of positional cloning projects. To date, most physical maps have been constructed by polymerase chain reaction (PCR)-based sequence-tagged site (STS) content mapping. While effective, this technique has a number of disadvantages including problems with yeast artificial chromosome (YAC) chimerism, the time and effort required to generate new STSs from YAC ends, the cost of primer synthesis for large contiging projects, and the time, effort, and expense necessary for screening each STS in the two-tiered hierarchical YAC library screening format. An alternative strategy, interspersed repetitive sequence (IRS) PCR genomics, alleviates many of these constraints. Clonal overlap is detected by hybridization of individual IRS-PCR products to IRS-PCR product pools of the three-dimensional coordinate pools of YAC libraries in dot-blot format. Entire libraries can be screened in a single step, and multiple libraries can be screened simultaneously. Cloning YAC fragments, sequencing, and primer generation are eliminated, increasing the efficiency of contig construction and reducing the expense. In addition, the genomic location of the individual IRS-PCR products can also be simultaneously determined by screening either interspecific backcrosses or radiation hybrid panels, in dot-blot format, confirming contig extension in the region of interest.

Animals↗

PubMed2000

Detection of hepatitis B virus infection in wild-born chimpanzees (Pan troglodytes verus): phylogenetic relationships with human and other primate genotypes.

Infection with hepatitis B virus (HBV) was detected by serological testing for HBV surface antigen and by PCR assay for HBV DNA in serum samples from two common chimpanzees (Pan troglodytes subsp. verus) born in West Africa. The complete genome sequences obtained by nucleotide sequencing of overlapping DNA fragments amplified by PCR were compared with HBV variants recovered from other primates and with human genotypes A to F. Both chimpanzee sequences were 3, 182 nucleotides in length, and the surface gene sequence predicted the existence of a, d, and w serological determinants. Neither sequence contained stop codons in the precore region. On phylogenetic analysis, the HBV variants infecting the chimpanzees clustered together with a third chimpanzee HBV isolate independently obtained from an infected captive animal (A. J. Zuckerman, A. Thornton, C. R. Howard, K. N. Tsiquaye, D. M. Jones, and M. R. Brambell, Lancet ii:652-654, 1978), with an overall sequence similarity of >94%. This provides strong evidence for a chimpanzee-specific genotype of HBV which circulates in nature. These findings add to the recent evidence for infection in the wild of other Old and New World primates (gibbon, orangutan, and woolly monkey) with species-specific variants of HBV. There is no evidence for close phylogenetic clustering of variants found so far in primates with any of the established HBV genotypes from humans. With the new evidence for the widespread distribution of HBV in primates, hypotheses for the origins of human infection are reviewed.

Animals↗

PubMed1996

Laser desorption mass spectrometry for point mutation detection.

A point mutation can be associated with the pathogenesis of inherited or acquired diseases. Laser desorption mass spectrometry coupled with allele specific polymerase chain reaction (PCR) was first used for point mutation detection. G551D is one of several mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene present in 1-3% of the mutant CFTR alleles in most European populations. In this work, two different approaches were pursued to detect G551D point mutation in the cystic fibrosis gene. The strategy is to amplify the desired region of DNA template by PCR using two primers that overlap one base at the site of the point mutation and which vary in size. If the two primers based on the normal sequence match the target DNA sequence, a normal PCR product will be produced. However, if the alternately sized primers that match the mutant sequence recognize the target DNA, an abnormal PCR product will be produced. Thus, the mass spectrometer can be used to identify patients that are homozygous normal, heterozygous for a mutation or homozygous abnormal at a mutation site. Another approach to identify similar mutations is the use of sequence specific restriction enzymes which respond to changes in the DNA sequence. Mass spectrometry is used to detect the length of the restriction fragments generated by digestion of a PCR generated target fragment.

Cell Line↗

PubMed1998

[Update on a diagnostic test for choroideremia: the protein truncation test (PTT)].

PURPOSE: The aim of this study was to define the RT-PCR-PTT parameters for CHM gene analysis and to evaluate its interest as a method for CHM mutation screening. METHODS: The entire CHM coding region was reversed-transcribed in three overlapping cDNA segments (RT-PCR) which were amplified and further analyzed by PTT after in vitro transcription/translation. RESULTS: This strategy enabled us to detect a truncated peptide in each of the 6 unrelated patients from southern France who were investigated. The mutation was further characterized by direct sequencing of the RT-PCR product. CONCLUSION: In CHM gene, all conditions are present to make the RT-PCR-PTT strategy the method of choice for mutation screening. As a result of the simplified protocol described in this study, the families of the patients could benefit from accurate carrier-status assessment.

Adaptor Proteins, Signal Transducing↗

PubMed1998

The rationale and method for constructing internal control DNA used in pertussis polymerase chain reaction.

The inclusion of an appropriate internal control DNA in polymerase chain reaction (PCR) is a rapid and simple method for the detection of PCR failure. Two PCR coamplification internal control DNAs (ICD I and ICD II) with the same primer-binding sequences as the target DNA for the detection of Bordetella pertussis and Bordetella parapertussis were produced using an overlap extension technique and a PCR MIMIC construction kit, respectively. The ICD II was further evaluated in a prospective clinical study in 360 patients with a clinical diagnosis of pertussis. From 360 nasopharyngeal swabs the internal control was positive in 318 (88%) samples, but was negative in 42 (12%). After phenol-chloroform extraction an additional 10 internal controls became positive. For the detection of PCR failure, the use of internal control DNA is highly recommended for PCR-based identification of B. pertussis and B. parapertussis organisms from nasopharyngeal swabs and aspirates.

Bordetella↗

PubMed1999

RT-PCR: characterization of long multi-gene operons and multiple transcript gene clusters in bacteria.

Reverse transcription (RT)-PCR is a valuable tool widely used for analysis of gene expression. In bacteria, RT-PCR is helpful beyond standard protocols of northern blot RNA/DNA hybridization (to identify transcripts) and primer extension (to locate their start points), as these methods have been difficult with transcripts that are low in abundance or unstable, similar to long multi-gene operons. In this report, RT-PCR is adapted to analyze transcripts that form long multi-gene operons--where they start and where they stop. The transcripts can also be semiquantitated to follow the expression of genes under different growth conditions. Examples using RT-PCR are presented with two different multi-gene systems for metal cation resistance to silver and mercury ions. The silver resistance system [9 open reading frames (ORFs); 12.5 kb] is shown by RT-PCR to synthesize three nonoverlapping messenger RNAs that are transcribed divergently. In the mercury resistance system (8 ORFs; 6.3 kb), all the genes are transcribed in the same orientation, and two promoter sites produce overlapping transcripts. For RT-PCR, reverse transcriptase enzyme is used to synthesize first-strand cDNA that is used as a template for PCR amplification of single-gene products, from the beginning, middle or end of long multi-gene, multi-transcript gene clusters.

Bacillus cereus↗

PubMed2004

The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by a defective oxidative burst of phagocytes and subsequent impairment of their microbicidal activity. Mutations in one of the NADPH-oxidase components affect gene expression or function of this system, leading to the phenotype of CGD. Defects in gp91-phox lead to X-linked CGD, responsible for approximately 70% of CGD cases. Investigation of the highly heterogeneous genotype of CGD patients includes mutation analysis, Northern blot or Western blot assays according to the particular case. The aim of the present study was to use reverse transcription (RT)-PCR for the analysis of molecular defects responsible for X-linked CGD in eight Brazilian patients and to assess its potential for broader application to molecular screening in CGD. Total RNA was prepared from Epstein B virus-transformed B-lymphocytes and reverse transcribed using random hexamers. The resulting cDNA was PCR-amplified by specific and overlapping pairs of primers designed to amplify three regions of the gp91-phox gene: exons 1-5, 3-9, and 7-13. This strategy detected defective gp91-phox expression in seven patients. The RT-PCR results matched clinical history, biochemical data (nitroblue tetrazolium or superoxide release assay) and available mutation analysis in four cases. In three additional cases, RT-PCR results matched clinical history and biochemical data. In another case, RT-PCR was normal despite a clinical history compatible with CGD and defective respiratory burst. We conclude that this new application of RT-PCR analysis--a simple, economical and rapid method--was appropriate for screening molecular defects in 7 of 8 X-linked CGD patients.

Child↗

PubMed2004

A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences.

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are approximately 500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Compared with the previously published methods, the PTDS method is rapid (5-7 days) and suitable for synthesizing long segments of DNA (5-6 kb) with high G + C contents, repetitive sequences or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures. Using the newly developed PTDS method, we have successfully obtained several genes of interest with sizes ranging from 1.0 to 5.4 kb.

Bacterial Proteins↗

PubMed2001

A powerful and rapid approach to human genome scanning using small quantities of genomic DNA.

Dense maps of short-tandem-repeat polymorphisms (STRPs) have allowed genome-wide searches for genes involved in a great variety of diseases with genetic influences, including common complex diseases. Generally for this purpose, marker sets with a 10 cM spacing are genotyped in hundreds of individuals. We have performed power simulations to estimate the maximum possible intermarker distance that still allows for sufficient power. In this paper we further report on modifications of previously published protocols, resulting in a powerful screening set containing 229 STRPs with an average spacing of 18.3 cM. A complete genome scan using our protocol requires only 80 multiplex PCR reactions which are all carried out using one set of conditions and which do not contain overlapping marker allele sizes. The multiplex PCR reactions are grouped by sets of chromosomes, which enables on-line statistical analysis of a set of chromosomes, as sets of chromosomes are being genotyped. A genome scan following this modified protocol can be performed using a maximum amount of 2.5 micrograms of genomic DNA per individual, isolated from either blood or from mouth swabs.

Alleles↗

PubMed1995

Detection of sequence variants in the gene encoding the beta 3 chain of laminin 5 (LAMB3).

Laminin 5, a candidate gene/protein system for mutations in the junctional forms of epidermolysis bullosa (JEB), consists of three polypeptides encoded by the LAMA3, LAMB3, and LAMC2 genes. In this study, primer pairs for the amplification of the complete cDNA as well as 22 exons of the LAMB3 gene encoding the entire beta 3 chain of laminin 5, were established. The primers for amplification of individual exons from genomic DNA were placed at least 50 bp away from the exon-intron borders in the flanking intronic sequences. For amplification of cDNA generated by RT-PCR, eight primer pairs covering overlapping segments of mRNA were used. The amplified sequences were used to study sequence variations of the LAMB3 gene in patients with JEB and unrelated individuals using heteroduplex analysis. Nine out of 13 JEB patients examined showed heteroduplexes in at least one of the PCR products, indicating the existence of two variable alleles in their DNA. Sequence analyses revealed putative pathogenetic mutations in seven of the JEB patients, while four of the heteroduplexes resulted from polymorphisms, reflecting a single basepair substitution. The results demonstrate that this method is useful in the detection of JEB mutations, as well as polymorphisms in the LAMB3 gene.

Base Sequence↗