Biomedical subjects

P J Oefner

Publications and source records attributed to P J Oefner.

At least 37 records · Page 2Linked to original sources

PubMed2000

Y chromosome sequence variation and the history of human populations.

Binary polymorphisms associated with the non-recombining region of the human Y chromosome (NRY) preserve the paternal genetic legacy of our species that has persisted to the present, permitting inference of human evolution, population affinity and demographic history. We used denaturing high-performance liquid chromatography (DHPLC; ref. 2) to identify 160 of the 166 bi-allelic and 1 tri-allelic site that formed a parsimonious genealogy of 116 haplotypes, several of which display distinct population affinities based on the analysis of 1062 globally representative individuals. A minority of contemporary East Africans and Khoisan represent the descendants of the most ancestral patrilineages of anatomically modern humans that left Africa between 35,000 and 89,000 years ago.

Africa↗

PubMed2000

A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis.

We developed a modified allele-specific PCR procedure for assaying single nucleotide polymorphisms (SNPs) and used the procedure (called SNAP for single-nucleotide amplified polymorphisms) to generate 62 Arabidopsis mapping markers. SNAP primers contain a single base pair mismatch within three nucleotides from the 3' end of one allele (the specific allele) and in addition have a 3' mismatch with the nonspecific allele. A computer program called SNAPER was used to facilitate the design of primers that generate at least a 1,000-fold difference in the quantity of the amplification products from the specific and nonspecific SNP alleles. Because SNAP markers can be readily assayed by electrophoresis on standard agarose gels and because a public database of over 25,000 SNPs is available between the Arabidopsis Columbia and Landsberg erecta ecotypes, the SNAP method greatly facilitates the map-based cloning of Arabidopsis genes defined by a mutant phenotype.

Alleles↗

PubMed2000

Cloning of the Arabidopsis RSF1 gene by using a mapping strategy based on high-density DNA arrays and denaturing high-performance liquid chromatography.

Mapping genes by chromosome walking is a widely used technique applicable to cloning virtually any gene that is identifiable by mutagenesis. We isolated the gene responsible for the recessive mutation rsf1 (for reduced sensitivity to far-red light) in the Arabidopsis Columbia accession by using classical genetic analysis and two recently developed technologies: genotyping high-density oligonucleotide DNA array and denaturing high-performance liquid chromatography (DHPLC). The Arabidopsis AT412 genotyping array and 32 F(2) plants were used to map the rsf1 mutation close to the top of chromosome 1 to an interval of approximately 500 kb. Using DHPLC, we found and genotyped additional markers for fine mapping, shortening the interval to approximately 50 kb. The mutant gene was directly identified by DHPLC by comparing amplicons generated separately from the rsf1 mutant and the parent strain Columbia. DHPLC analysis yielded polymorphic profiles in two overlapping polymorphic amplicons attributable to a 13-bp deletion in the third of five exons of a gene encoding a 292-amino acid protein with a basic helix-loop-helix (bHLH) domain. The mutation in rsf1 results in a truncated protein consisting of the first 129 amino acids but lacking the bHLH domain. Cloning the RSF1 gene strongly suggests that numerous phytochrome A-mediated responses require a bHLH class transcription factor.

Alleles↗

PubMed2000

High-accuracy DNA sequence variation screening by DHPLC.

Genetic maps based on biallelic single-nucleotide polymorphisms amenable to microarray-based genotyping have significantly accelerated the mapping of mono- and multigenic traits in model organisms such as Saccharomyces cerevisiae and Arabidopsis thaliana. This advance needs to be matched by highly accurate, inexpensive and robust methodology for fine-structure mapping of the candidate region(s) and the eventual identification of the causative mutation(s). To establish the usefulness of denaturing high-performance liquid chromatography (DHPLC) for those purposes, we have amplified 476 fragments from two A. thaliana ecotypes with an average length of 563 bp covering various candidate regions on chromosomes 1, 2 and 4. Parallel analysis by DHPLC and dye terminator sequencing showed that DHPLC detected 165 out of 166 polymorphic fragments with only four false positives, amounting to a sensitivity, specificity and accuracy of 99.4%, 98.7% and 99%, respectively. It proved beneficial to analyze the fragments not only at the highest but also at the lower temperatures recommended by the algorithm freely available at http:¿insertion.stanford.edu/melt.html.

Algorithms↗

PubMed1999

Distribution of haplotypes from a chromosome 21 region distinguishes multiple prehistoric human migrations.

Despite mounting genetic evidence implicating a recent origin of modern humans, the elucidation of early migratory gene-flow episodes remains incomplete. Geographic distribution of haplotypes may show traces of ancestral migrations. However, such evolutionary signatures can be erased easily by recombination and mutational perturbations. A 565-bp chromosome 21 region near the MX1 gene, which contains nine sites frequently polymorphic in human populations, has been found. It is unaffected by recombination and recurrent mutation and thus reflects only migratory history, genetic drift, and possibly selection. Geographic distribution of contemporary haplotypes implies distinctive prehistoric human migrations: one to Oceania, one to Asia and subsequently to America, and a third one predominantly to Europe. The findings with chromosome 21 are confirmed by independent evidence from a Y chromosome phylogeny. Loci of this type will help to decipher the evolutionary history of modern humans.

Animals↗

PubMed1999

Identification by denaturing high-performance liquid chromatography of numerous polymorphisms in a candidate region for multiple sclerosis susceptibility.

Genetic association analysis of candidate regions where evidence of linkage has accumulated is becoming a key issue in the study of complex diseases. A high density of markers, at least one per centimorgan, is required to improve the chances of observing linkage disequilibrium with disease alleles. A recently available single nucleotide polymorphism (SNP) map designed to cover the whole genome provides an average density of one marker per 2 cM. In the present study we show that the number of markers can be approximately doubled in a selected region, thus reaching a density suitable for association studies, by applying a completely automated technique for polymorphism detection, denaturing high-performance liquid chromatography (DHPLC). A systematic search for SNPs was performed in the region 5ptel-q13, where weak but convergent evidence for linkage with multiple sclerosis has accumulated. Screening for polymorphisms was performed on 124 sequence tagged sites (STSs) in the 3'UTR ends of expressed sequence tags totaling about 30,000 bp. Thirty SNPs in 28 STSs were found with less than 10% overlap with the markers already detected in the same region. The data confirm the validity of the approach using DHPLC on expressed gene sequences tagged by a set of standard commercially available primers.

Chromatography, High Pressure Liquid↗

PubMed1999

Genotyping single nucleotide polymorphisms by primer extension and high performance liquid chromatography.

We have investigated the possibility of genotyping single nucleotide polymorphisms (SNPs) by primer extension and high performance liquid chromatography (HPLC). Using three polymorphisms of current interest to our group (an A/G polymorphism in the proneurotensin gene and A/G and T/C polymorphisms in the 5HT2a receptor gene), we show that robust signal is obtained using this simple analytic method which has the added advantages that sample loading and analysis are essentially automated, analytic time is brief, and no further purification step after primer extension is required. We also show that all stages of the HPLC-primer extension genotyping can be multiplexed which, together with automation, suggests that this system may be suitable for linkage studies based upon emerging SNP maps.

Base Sequence↗

PubMed1999

Genome-wide mapping with biallelic markers in Arabidopsis thaliana.

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

Arabidopsis↗

PubMed1999

Altered sodium pump alpha and gamma subunit gene expression in nephron segments from hypertensive rats.

OBJECTIVE: To determine the qualitative and quantitative expression of alpha and gamma sodium pump subunits in whole kidney and nephron segment RNA from Sprague Dawley rats, spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. DESIGN: A novel reverse transcription polymerase chain reaction technique was devised which provides accurate and precise measurement of the number of molecules of specific transcript abundance, a measurement of gene expression. This allows the quantitative comparison of multiple samples across multiple subjects and, since the estimates are accurate rather than relative, can also be used to make quantitative comparisons across expressed genes, such as isoforms and subunits of the heterotrimeric renal sodium pump. METHODS: We examined which catalytic isoforms were expressed and then quantified transcript abundance in whole kidney and convoluted and straight segments of the proximal tubule. RESULTS: Alpha 1 and gamma transcripts, but not alpha 2, alpha 3 or alpha 4 isoforms, were consistently observed in nephron segments. Levels of alpha 1 were lower in kidney RNA from 15-16-week-old SHR than in WKY rats of the same age (P = 0.001), but were not different between SHR and WKY in 4-5-week-old animals. No significant difference was observed in gamma subunit abundance in kidney RNA from 4-5-week-old animals; however, at 15-16 weeks, the expression in SHR was one-third that in WKY rats (P = 0.003). In proximal convoluted tubules from 4-5-week-old animals, the level of alpha 1 RNA expression was lower (P = 0.03) in SHR than in WKY rats. In addition, levels of alpha 1 in proximal straight tubule from the 4-5-week-old SHR were also lower than in WKY rats (P = 0.02). This difference was even greater in 15-16-week-old animals: in SHR, alpha 1 expression was less than 20% of the level of expression in WKY rats (P = 0.0003). Expression of the gamma subunit exhibited a similar pattern of downregulation in SHR. In RNA from proximal convoluted tubules and proximal straight tubules from both 4-5- and 15-16-week-old animals, expression of the gamma subunit was demonstrated to be significantly lower in SHR than in WKY rats. CONCLUSION: The results indicate a coordinate reduction in the abundance of sodium pump alpha and gamma subunits in the proximal tubules of SHR, which occurs early during the development of hypertension.

Animals↗

PubMed1999

Optimal temperature selection for mutation detection by denaturing HPLC and comparison to single-stranded conformation polymorphism and heteroduplex analysis.

BACKGROUND: Denaturing HPLC (DHPLC) is a semi-automated method for detecting unknown DNA sequence variants. The sensitivity of the method is dependent on the temperature at which the analysis is undertaken, the selection of which is dependent on operator experience. To circumvent this, software has been developed for predicting the optimal temperature for DHPLC analysis. We examined the utility of this software. METHODS: To maximize the relevance of our data for other investigators, we have screened 42 different amplimers from CFTR, TSC1, and TSC2. The samples consisted of 103 unique sequence heterozygotes and 126 wild-type homozygous controls. RESULTS: At the temperature recommended by the software, 96% (99 of 103) of heterozygotes and all of the wild-type controls were correctly classified. This compares favorably with sensitivities of 85% for single-stranded conformation polymorphism and 82% for gel-based heteroduplex analyses of the same fragments. CONCLUSIONS: Software-optimized DHPLC is a highly sensitive method for mutation detection. However, where sensitivity >96% is required, our data suggest that in addition to the recommended temperature, fragments should also be run at the recommended temperature plus 2 degrees C.

Chromatography, High Pressure Liquid↗

PubMed1998

Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection.

Denaturing high-performance liquid chromatography (DHPLC) is a novel high-capacity technique for detecting new mutations. We have evaluated the sensitivity and specificity of this method in a blind analysis of exon H of the factor IX gene and exon 16 of the neurofibromatosis type 1 gene. Under a single set of conditions for each exon, 55/55 individuals carrying 48 unique mutations were correctly identified as were 55/55 individuals with wildtype alleles. We conclude that DHPLC is a highly sensitive and specific method for mutation detection.

Chromatography, High Pressure Liquid↗

PubMed1998

Modeling and analysis of competitive RT-PCR.

The present studies demonstrate a theoretical and practical framework for the accurate quantitation of gene expression in RNA extracted from microscopic tissue samples. The approaches are developed around competitive RT-PCR techniques. Assay performance has been examined and validated at both the RT and PCR steps. Our analysis of RT transcription efficiency for a number of native and competitor combinations shows that this property can differ, even for very similar templates. However, this difference is consistent and, once identified and measured, can be removed as an obstacle to accuracy. Using mathematical modeling, we have examined the simulated co-amplification of native and competitor templates in PCR. Useful insights have emerged from such modeling which indicate that differences in initial amplification efficiency and the rate of decay of amplification efficiency during the reaction can rapidly lead to inaccuracy, even while the slope and linearity of log plots of the competitor input and reaction product ratios are close to ideal. Finally, we show here that competitive RT-PCR reactions do not have to remain in the log-linear phase of PCR in order to accomplish accurate and precise quantification. Using appropriate competitors sharing primer binding sites and high internal sequence similarity, identical amplification efficiencies are preserved throughout the reaction. Reaction products, including heteroduplexes formed between native and competitor templates as reactions progress to plateau, can be identified and quantified accurately using the new technique of denaturing HPLC (dHPLC). This analytical technique allows the accuracy of competitive RT-PCR to be preserved beyond the linear phase. The technique has high sensitivity and precision and target abundances as low as 100 copies could be reliably estimated.

Animals↗

PubMed1998

Quantitative analysis of gene expression by ion-pair high-performance liquid chromatography.

We have analyzed the utility of ion-pair reversed-phase HPLC for gene quantification by competitive reverse transcriptase polymerase chain reaction (RT-PCR). Competitive RT-PCR reactions employed various RNA competitors which shared high sequence similarity to the native transcripts for which they served as references. Competitive reactions resulted in the detection of two reaction products when reactions were analyzed by agarose gel electrophoresis, but three products when analyzed by HPLC. The third product was demonstrated to be a heteroduplex formed between mixed strands of native and competitor amplicons. Mathematical analysis of these competitive reactions indicated that identification and quantification of the heteroduplexes were essential to produce accurate gene quantification. PCR amplification efficiency was shown to be identical for native and competitor transcripts. However, RT efficiency differences were observed which may be sequence dependent. These differences were highly consistent across reactions for the same native and competitor inputs. Increasing the sequence similarity resulted in a competitor which had the same RT efficiency as the native transcript. Titration of various levels of competitor against native RNA resulted in the expected linear relationships which had slopes of unity. Quantitation could be performed with similar precision in single tube comparisons in which the initial abundance of the native transcript was calculated by knowledge of the final reaction product ratio and the initial competitor input level. The assay system is highly accurate, i.e. the measured level of gene expression reflected the actual copy number of the gene present in the sample. This was demonstrated by performing reactions in which known amounts of native transcript were quantified and the amount estimated by the assay was shown to be the same as the known amount added to the reaction. A similar approach has been devised for examining the relative levels of alternatively spliced isoforms. In this system, primers were selected to produce reaction products which served as their own internal competitors (by spanning the alternative splice site). Hormonal dependence of the ratio of abundance of two isoforms of the rabbit RUSH-1 gene was demonstrated.

Alternative Splicing↗

PubMed1998

An automated hydrodynamic process for controlled, unbiased DNA shearing.

An automated, inexpensive, easy-to-use, and reproducible technique for controlled, random DNA fragmentation has been developed. The technique is based on point-sink hydrodynamics that result when a DNA sample is forced through a small hole by a syringe pump. Commercially available components are used to reduce the cost and complexity of the instrument. The design is optimized to reduce the volume of sample required and to speed processing time. Shearing of the samples can be completely automated by computer control. Ninety percent of sheared DNA fragments fall within a twofold size distribution that is highly reproducible. Three parameters are critical: the flow geometry, the flow rate, and a minimum number of iterations. Shearing is reproducible over a wide range of temperatures, DNA concentrations, and initial DNA size. The cloning efficiency of the sheared DNA is very good even without end repair, the distribution of assembled sequences is random, and there is no sequence bias at the ends of sheared fragments that have been cloned. The instrument, called the Point-sink Shearer (PtS), has already been exported successfully to many other laboratories.

Automation↗

PubMed1998

Analyses of the NRAMP1 and IFN-gammaR1 genes in women with Mycobacterium avium-intracellulare pulmonary disease.

Mycobacterium avium-intracellulare (MAI) pulmonary disease causes substantial morbidity in a population of older, HIV-negative women without preexisting lung disease. The cause for disease susceptibility in these patients is unknown, although their relative phenotypic homogeneity suggests the existence of a common, subtle immune deficiency. An investigation was undertaken to determine if these patients have a defect in their natural resistance-associated macrophage protein (NRAMP1) or interferon gamma receptor 1 (IFN-gammaR1) genes. A point mutation in murine nramp, an autosomal recessive gene controlling resistance to intracellular organisms, correlates with overwhelming Mycobacterium bovis infection in mice. The corresponding region in human NRAMP1, two coding polymorphisms and one promoter NRAMP1 polymorphism, as well as two IFN-gammaR1 polymorphisms, were analyzed to determine if an allele was present to correlate with disease. Genomic DNA was purified from eight women with MAI pulmonary disease and four controls. Regions of interest were amplified by PCR; three sites were analyzed by restriction fragment length polymorphisms, and three were analyzed using denaturing high-performance liquid chromatography. The NRAMP1 promoter polymorphism of 18 additional random controls was analyzed by microsatellite sizing. No allelism was found in NRAMP1 corresponding to the murine mutation, or in the two coding regions. In the NRAMP1 promoter microsatellite, 3 of 8 patients were heterozygous for a dinucleotide sequence insertion, as were 10 of 22 controls. None of the patients had either of the two known IFN-gammaR1 mutations. In conclusion, in women with MAI pulmonary disease, there is no evidence for a genetic defect in NRAMP1 or IFN-gammaR1 to correlate with disease.

Aged↗

PubMed1997

Quantification of alternatively spliced RUSH mRNA isoforms by QRT-PCR and IP-RP-HPLC analysis: a new approach to measuring regulated splicing efficiency.

Quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) and the ion-pair reverse-phase (IP-RP)-HPLC product purification and detection system were developed to facilitate the isolation and proportional quantification of alternatively spliced RUSH mRNAs. RUSH isoforms result from alternative splicing of a 57-bp exon and encode SNF/SWI-related proteins that bind to the uteroglobin promoter. QRT-PCR was performed using total RNA, and a pair of primers designed to flank the 57-bp exon. When more than one splice variant was expressed, IP-RP-HPLC identified the specific homoduplex products, as well as the heteroduplexes formed as a consequence of partial sequence complementarity between the products. Data analysis included the correct re-allocation of heteroduplex components to achieve accurate quantitation of changes in the relative levels of RUSH message isoforms. The preferential expression of the RUSH-1alpha isoform by all the tissues except estrous uterine endometrium and lactating mammary gland indicates RUSH pre-mRNAs are alternatively spliced in a tissue-specific manner. A 61-fold difference in the relative rate of RUSH pre-mRNA splicing is indicated by the difference in the ratios of RUSH mRNA isoforms from uterine endometrium and testis. Clearly, QRT-PCR and IP-RP-HPLC are powerful and versatile tools for the detection and quantitation of mRNA splice variants.

Alternative Splicing↗

PubMed1997

Detection of numerous Y chromosome biallelic polymorphisms by denaturing high-performance liquid chromatography.

Y chromosome haplotypes are particularly useful in deciphering human evolutionary history because they accentuate the effects of drift, migration, and range expansion. Significant acceleration of Y biallelic marker discovery and subsequent typing involving heteroduplex detection has been achieved by implementing an innovative and cost-efficient method called denaturing high-performance liquid chromatography (DHPLC). The power of the method resides in its sensitivity and ability to rapidly compare amplified sequences in an automated manner. We have determined the allelic states of 22 Y polymorphisms; 19 of which are unreported, in 718 diverse extant chromosomes; established haplotype frequencies; and deduced a phylogeny. All major geographic regions, including Eurasia, are characterized by mutations reflecting episodes of genetic drift and expansion. Most biallelic markers are localized regionally. However, some show wider dispersal and designate older, core haplotypes. One transversion defines a major haplogroup that distinguishes a previously unknown deep, apparently non-African branch. It provides evidence of an ancient bottleneck event. It is now possible to anticipate the inevitable detailed reconstruction of human Y chromosome genealogy based on several tens to even hundreds of these important polymorphisms.

Alleles↗

PubMed1997

Hepatitis G virus infection in hemodialysis patients and the effects of interferon treatment.

OBJECTIVE: To characterize the nature of hepatitis G virus (HGV) infections in hemodialysis patients and to determine the responsiveness of HGV to antiviral therapy in these patients. METHODS: HGV, a recently identified flavivirus, is associated with non-A-E viral hepatitis infections. We studied HGV infections in hepatitis C virus (HCV)-infected hemodialysis patients over a 1-yr period, using two independent PCR assays and nucleic acid sequencing. Thirty-four of 63 study patients were treated with interferon. RESULTS: We observed a 27% prevalence (17/63 patients) and a 4% annual incidence of HGV infections in the study population. HGV was not detected in any of the 10 HGV-infected patients immediately after interferon therapy. Although seven of these 10 patients developed HGV relapses, three had long-term responses. The interferon responsiveness of HGV and HCV appeared to be unrelated. In contrast, all seven untreated HGV-infected patients remained viremic. Sequence analyses of the different HGV isolates revealed only very limited genetic variability in the polymerase chain reaction-amplified regions of HGV during 1 yr of observation. CONCLUSIONS: Our data suggest that HCV-infected hemodialysis patients are at substantial risk of acquiring HGV infection and that HGV infections are prevalent in this population. In addition, HGV infections become chronic but are responsive to interferon treatment.

Adult↗