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G Ruano

Publications and source records attributed to G Ruano.

At least 19 recordsLinked to original sources
PubMed2001

Haplotype variation and linkage disequilibrium in 313 human genes.

Variation within genes has important implications for all biological traits. We identified 3899 single nucleotide polymorphisms (SNPs) that were present within 313 genes from 82 unrelated individuals of diverse ancestry, and we organized the SNPs into 4304 different haplotypes. Each gene had several variable SNPs and haplotypes that were present in all populations, as well as a number that were population-specific. Pairs of SNPs exhibited variability in the degree of linkage disequilibrium that was a function of their location within a gene, distance from each other, population distribution, and population frequency. Haplotypes generally had more information content (heterozygosity) than did individual SNPs. Our analysis of the pattern of variation strongly supports the recent expansion of the human population.

Alleles↗
PubMed2000

Complex promoter and coding region beta 2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness.

The human beta(2)-adrenergic receptor gene has multiple single-nucleotide polymorphisms (SNPs), but the relevance of chromosomally phased SNPs (haplotypes) is not known. The phylogeny and the in vitro and in vivo consequences of variations in the 5' upstream and ORF were delineated in a multiethnic reference population and an asthmatic cohort. Thirteen SNPs were found organized into 12 haplotypes out of the theoretically possible 8,192 combinations. Deep divergence in the distribution of some haplotypes was noted in Caucasian, African-American, Asian, and Hispanic-Latino ethnic groups with >20-fold differences among the frequencies of the four major haplotypes. The relevance of the five most common beta(2)-adrenergic receptor haplotype pairs was determined in vivo by assessing the bronchodilator response to beta agonist in asthmatics. Mean responses by haplotype pair varied by >2-fold, and response was significantly related to the haplotype pair (P = 0.007) but not to individual SNPs. Expression vectors representing two of the haplotypes differing at eight of the SNP loci and associated with divergent in vivo responsiveness to agonist were used to transfect HEK293 cells. beta(2)-adrenergic receptor mRNA levels and receptor density in cells transfected with the haplotype associated with the greater physiologic response were approximately 50% greater than those transfected with the lower response haplotype. The results indicate that the unique interactions of multiple SNPs within a haplotype ultimately can affect biologic and therapeutic phenotype and that individual SNPs may have poor predictive power as pharmacogenetic loci.

Base Sequence↗
PubMed2000

The accuracy of statistical methods for estimation of haplotype frequencies: an example from the CD4 locus.

Haplotype analysis has become increasingly important for the study of human disease as well as for reconstruction of human population histories. Computer programs have been developed to estimate haplotype frequencies statistically from marker phenotypes in unrelated individuals. However, there currently are few empirical reports on the accuracy of statistical estimates that must infer linkage phase. We have analyzed haplotypes at the CD4 locus on chromosome 12 that consist of a short tandem-repeat polymorphism and an Alu insertion/deletion polymorphism located 9.8 kb apart, in 398 individuals from 10 geographically diverse sub-Saharan African populations. Haplotype frequency estimates obtained using gene counting based on molecularly haplotyped (phase-known) data were compared with haplotype frequency estimates obtained using the expectation-maximization algorithm. We show that the estimated frequencies of common haplotypes do not differ significantly with the use of phase-known versus phase-unknown data. However, rare haplotypes are occasionally miscalled when their presence/absence must be inferred. Thus, for those research questions for which the common haplotypes are most important, frequency estimates based on the phase-unknown marker-typing results from unrelated individuals will be sufficient. However, in cases where knowledge of rare haplotypes is critical, molecular haplotyping will be necessary to determine linkage phase unambiguously.

Africa South of the Sahara↗
PubMed1996

Molecular haplotyping of genetic markers 10 kb apart by allele-specific long-range PCR.

Haplotypes, combinations of polymorphic markers in a chromosome, are critical for genome diversity research. However, their utility in population samplings is compromised by uncertain linkage phase determinations from unrelated individuals. Molecular haplotyping accomplishes direct phase determination by generation of hemizygous templates from diploid genomic samples. We report molecular haplotyping by allele-specific long-range PCR of two markers 9.5 kb apart at the CD4 locus: a bi-allelic Alu deletion and a multi-allelic repeat. We verified CD4 molecular haplotypes by classical Mendelian analysis. Molecular haplotyping should prove useful in mapping disease genes and in establishing founder effects.

Alleles↗
PubMed1996

Distribution and frequency of a polymorphic Alu insertion at the plasminogen activator locus in humans.

We have investigated the frequency distribution, across a broad range of geographically dispersed populations, of alleles of the polymorphic Alu insertion that occurs within the 8th intron of the tissue plasminogen, activator gene (PLAT). This Alu is a member of a recently derived subfamily of Alu elements that has been expanding during human evolution and continues to be transpositionally active. We used a "population tube" approach to screen 10 chromosomes from each of 19 human populations for presence or absence of this Alu in the PLAT locus and found that all tested populations are dimorphic for presence/absence of this insertion. We show that the previously published EcoRI, HincII, PstI, TaqI, and XmnI polymorphisms at the PLAT locus all result from insertion of this Alu and we use both restriction fragment length polymorphism and polymerase chain reaction analysis to examine the frequency of Alu(+) and Alu(-) alleles in a sample of 1003 individuals from 27 human populations and in 38 nonhuman primates. Nonhuman primates are monomorphic for the Alu(-) allele. Human populations differ substantially in allele frequency, and in several populations both alleles are common. Our results date the insertion event prior to the spread and diversification of modern humans.

Base Sequence↗
PubMed1995

Human genomic characterization of a novel locus-specific repetitive sequence.

A novel human chromosome locus-specific repetitive sequence was identified and characterized using arbitrary PCR. The repeat monomer consensus sequence is 100 bp long, and there are a minimum of 140 to 160 copies of the repetitive sequence per haploid human genome. The repetitive sequence is highly clustered on 20q12 within a 200- to 400-kb region. The highly polymorphic repeat array is inherited in a stable Mendelian fashion. Hybridization analysis revealed detectable conservation of the repeated element only among hominoids and Old World monkeys, where repeat arrangements are also polymorphic.

Animals↗
PubMed1994

Detection of DNA sequence variation via deliberate heteroduplex formation from genomic DNAs amplified en masse in "population tubes".

We have developed the population tube (poptube) system for sensitive detection and large-scale sampling of DNA sequence variation in several human populations of wide geographic distribution. In this methodology, genomic DNAs from five individuals in a population are PCR amplified en masse to maximize deliberately the chances of forming heteroduplexes among allelic variants. Interpopulation mixing is performed in a separate set of tubes containing one individual from each of five populations as well as a reference chimpanzee sample deliberately chosen to be different from all humans. Mismatches at sites of allelic variation retard the electrophoresis and reduce the stability of heteroduplex molecules. The products are electrophoresed on denaturing gradient gels where detection of heteroduplexes is accomplished readily. Using poptubes, we have discovered a rare variant in an otherwise highly conserved 440-bp segment in the long intron of the glucose-6-phosphate dehydrogenase (G6PD) gene. The polymorphism at this X-chromosome locus could be only detected in males by mixing samples, as homoduplexes for both alleles co-focus on denaturing gradient electrophoresis.

Animals↗
PubMed1993

Cycled primer extension: a method for DNA amplification and labeling from templates of unknown sequence.

A method has been developed for simultaneous radiolabeling and amplification of DNA hybridization probes. The method is termed cycled primer extension (CPE). CPE is a series of temperature-driven reactions in which template DNA is successively denatured and extended by a thermostable primer-dependent DNA polymerase. The primers consist of semirandom nanomers of the form 5'-NNN NNN (G/C)(G/C)(G/C)-3'. These nanomers have the capacity to anneal to any template DNA and serve as initial anchors for extension at the high temperatures required for Taq DNA polymerase activity. CPE cycles consist of 94 degrees C denaturation, annealing of primers to template upon ramping to 24 degrees C, and gradual extension of the primer along the template as temperature is ramped back to 94 degrees C. Labeling efficiency with [32P]dCTP was examined and optimized as determined by the relation to ratios of radiolabeled to unlabeled dCTP, by number of cycles, and by primer composition and sequence. CPE probes can be generated without regard to size or sequence of template and have a high specific activity (approximately 10(9) dpm/micrograms). With CPE, hybridization signals equivalent to those from random primed probes are routinely obtained with initial template amounts as low as 1 ng.

DNA↗
PubMed1992

DNA sequence polymorphism within hominoid species exceeds the number of phylogenetically informative characters for a HOX2 locus.

Within- and between-species variability was examined in a noncoding 238-bp segment of the HOX2 cluster. DNA of 4-26 individuals of four species (Pongo pygmaeus, Pan troglodytes, Gorilla gorilla, and Homo sapiens) was PCR amplified and electrophoresed in a denaturing gradient gel to screen for variability. Coupled amplification and sequencing was used to determine the complete sequence for each of the different alleles identified, one each in humans and orangutans, two in chimpanzees, and four in gorillas. Maximum-parsimony methods were used to construct a gene tree for these sequences. Alleles in all four species cluster into groups consisting of only one species (i.e., alleles within a species are monophyletic). The number of base-pair differences observed among alleles within P. troglodytes and within G. gorilla is larger than the number of base-pair substitutions that phylogenetically link Pan with Homo. Given these and other published data, it is premature to accept any particular phylogenetic tree that relates these three genera through two separate speciation events.

Animals↗
PubMed1992

Modeling of heteroduplex formation during PCR from mixtures of DNA templates.

We have investigated the capability of PCR to amplify a specific locus from each template in a mixture of allelic DNAs. Modeling experiments employed a 300-bp HOX2B segment as target and utilized, as distinct template "alleles," genomic DNAs from 1 human and from 2 chimpanzees known to differ in sequence at the target. Two modes were used: (1) mixtures of PCR products that had been previously amplified individually from a single template and (2) PCR amplification en masse from composite human/chimpanzee genomic DNA templates. Products generated by either mode were separated by denaturing gradient gel electrophoresis (DGGE). Detection of a "trace" allele mixed with a "dominant" one was possible by simple ethidium bromide staining of the gel up to a sensitivity of 1 part in 20. A balanced mixture was represented by 5:5 and 4:3:3 mixtures of allelic PCR products or genomic templates; an uneven mixture of dominant and trace alleles, by 9:1 and 8:1:1 mixtures. For 5:5, two homoduplex bands and two heteroduplex bands of equal intensity were generated. For 9:1, the trace homoduplex disappears whereas the two heteroduplexes are easily visible. For 8:1:1, four heteroduplexes and one homoduplex were observed; homoduplexes and heteroduplexes formed from the trace alleles were not visible. These experiments demonstrate that PCR can amplify mixed allelic templates in direct proportion to the stoichiometric fraction of each template. Trace species are captured as heteroduplexes with the most abundant species and are clearly displaced on denaturing gradient gels from the dominant homoduplex species. Our analytical studies can be applied to analysis of sequence variants in DNA collected from cancerous or infected tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Heat-soaked PCR: an efficient method for DNA amplification with applications to forensic analysis.

Heat-soaked PCR (HS-PCR) is a method for enhancing amplification performed by heating the DNA sample at 94 degrees C in 90 microliters 1.1 x buffer for 30 min. A 10-microliters bolus of concentrated (10x) deoxynucleotides, Taq DNA polymerase and primers prepared without buffer is then added just prior to thermal cycling. We have investigated the application of this method in a variety of forensically important DNA samples and compared it with regular PCR (R-PCR). DNA samples extracted from bone, postmortem tissues, bloodstains and hair contained low concentrations of human DNA or were contaminated with either non- human DNA or hemoglobin degradation products. Optimal conditions for HS-PCR were determined for the 3' ApoB VNTR locus and applied to a centromeric repeat element and to a single-copy locus. HS-PCR consistently and reproducibly enhanced product yield and specificity over R-PCR at all three loci in the entire set of DNA samples. HS-PCR was also effective in overcoming the inhibitory effect of hemoglobin at concentrations that fully impeded R-PCR.

DNA↗
PubMed1992

A polymerase chain reaction (PCR) method for sex and species determination with novel controls for deoxyribonucleic acid (DNA) template length.

Human X and Y chromosome alpha-satellite sequences lying within higher order repeats were amplified by the polymerase chain reaction (PCR) in genomic deoxyribonucleic acid (DNA) isolated from blood, bone, and several other tissues and specimens of potential forensic science interest. X and Y sequences could be coamplified under some of the PCR conditions employed. Monomorphic sequences in the 3'-apolipoprotein B gene (designated "H") and in an alpha-satellite higher order repeat on Chromosome 17 (p17H8, D17Z1) were likewise amplified in the specimens. X and Y sequence amplification can provide information about the sex of origin. Amplification of the X, H, and D17Z1 sequences was found to be primate-specific among the common animals tested and can thus provide species of origin information about a specimen. The authors suggest that amplification of X and D17Z1 or H sequences might provide "relaxed" and "stringent" controls for appropriate PCR amplification tests on forensic science specimens. Testing was carried out using PCR protocols that employed Thermophilus aquaticus (Taq) and Thermus flavis (Replinase) thermostable DNA polymerases.

Animals↗
PubMed1991

Genotyping and haplotyping of polymorphisms directly from genomic DNA via coupled amplification and sequencing (CAS).

Coupled amplification and sequencing (CAS) allows a segment of DNA to be sequenced directly from genomic DNA. An initial PCR amplification stage selects and amplifies the target. During a subsequent stage both strands of the target segment are sequenced simultaneously and amplified further. We show that CAS can readily identify variant base pairs. Genotyping of a population for known sequence variation can be achieved simply and directly from genomic DNA of each organism by performing CAS only for the variant bases. The procedure supercedes development and optimization of alternative typing assays based on oligonucleotide hybridization or ligation. In addition, we show that competitive oligonucleotide priming with allelic primers can be readily performed in concert with the second stage of CAS. The combination of techniques allows sequencing of a single chromosome from a heterozygous genomic sample and direct haplotyping of the polymorphism at the priming site with any others encompassed within the amplified segment.

Animals↗
PubMed1991

Coupled amplification and sequencing of genomic DNA.

Addition of dideoxyribonucleotides during the exponential phase of the PCR should result in the synthesis of two complementary sequence ladders. We have explored this hypothesis to develop coupled amplification and sequencing of genomic DNA. Coupled amplification and sequencing is a biphasic method for sequencing both strands of template as they are amplified. Stage I selects and amplifies a single target from the genomic DNA sample. Stage II accomplishes the sequencing as well as additional amplification of the target using aliquots from the stage I reaction mixed with end-labeled primer and dideoxynucleotides. We have successfully applied coupled amplification and sequencing to a 300-base-pair fragment 4 kilobases upstream from HOX2B directly from human whole genomic DNA.

Base Sequence↗
PubMed1991

Genetic markers in human bone: I. Deoxyribonucleic acid (DNA) analysis.

Deoxyribonucleic acid (DNA) was isolated from a number of spongy and compact human bone tissue specimens, and the yield was estimated on a "per milligram of starting tissue" basis. DNA was, in addition, isolated from a number of corresponding blood and bone tissue specimens. Spectrophotofluorometry and ethidium bromide visualization on minigels were used to estimate the quantity and degree of degradation of DNA. The DNA from several blood-bone pairs is shown to give concordant restriction fragment length polymorphism (RFLP) typing results by two different typing protocols with five different single-locus probes. DNA from several additional blood-bone pairs is shown to give concordant results for human leucocyte antigen (HLA)-DQ alpha phenotypes following polymerase chain reaction (PCR) amplification and hybridization to specific allele-specific oligonucleotide (ASO) probes, and for the variable numbers of tandem repeats (VNTR) length polymorphisms 3' to the human apolipoprotein B (APOB) gene following PCR amplification with specific primers and analysis of the products by electrophoresis and ethidium bromide visualization.

Base Sequence↗