Deoxyribonucleic Acid Markers Research Articles

Comparing Three DNA Extraction Procedures for Cost, Efficiency, and DNA Yield

An efficient deoxyribonucleic acid (DNA) extraction procedure that yields large quantities of DNA would provide adequate DNA for a large number of different analytical procedures. This study was conducted to compare three DNA extraction procedures for cost, time efficiency, and DNA content while extracting DNA from Kentucky bluegrass (Poa pratensis L.). Three students at the Univ. of Illinois with varying levels of DNA extraction experience conducted DNA extractions using Plant DNeasy™ Mini Kits, Plant DNAzol® Reagent, and a PEX/CTAB buffer. Costs varied significantly with cost (US$) per DNA sample of $3.04 for the DNeasy™ method, $0.99 for the DNAzol® method, and $0.39 for the PEX/CTAB extraction. The DNAzol® method was the fastest; although extracting 2.8 ng less DNA than the DNeasy™ method, it did not require the use of hazardous organic solvents, and random amplified polymorphic DNA (RAPD) markers were satisfactory for DNA fingerprinting of Kentucky bluegrass cultivars. The PEX/CTAB method, which did not include a tissue homogenization step, did not have reproducible banding patterns due to miniscule and inconsistent quantities of DNA extracted, or possibly due to inadequate purification. The investigator with the least DNA extraction experience was the slowest, while extracting 75% more DNA. All three methods are easily adapted to laboratories having personnel with different levels of experience. The DNAzol® Reagent method should save time and money, with reproducible results when many individual plant samples need to be identified. Chemical names used: potassium ethyl xanthogenate (PEX); cetyltrimethyl ammonium bromide (CTAB)

Triatomine vectors of Trypanosoma cruzi: a molecular perspective based on nuclear ribosomal DNA markers

Chagas disease (American trypanosomiasis) is mainly transmitted by blood-sucking bugs of the reduviid subfamily Triatominae (Hemiptera: Prosorrhyncha). Control strategies are directed mainly against these insect vectors, as no vaccine is available and, except in the very early stage of infection, there is no effective chemotherapy. Studies of deoxyribonucleic acid (DNA) will lead to major advances in our knowledge of Triatominae and their relationships to Chagas disease transmission, epidemiology and control. Analyses of complete sequences of nuclear genes coding for ribosomal ribonucleic acid (rRNA) ( rRNA genes) and spacers furnish significant information at the levels of higher taxons, genera, species, subspecies, hybrids, varieties and populations of Triatominae. This paper briefly reviews the contributions of studies on the slowly-evolving 18S or small subunit (SSU) rRNA gene and the quickly-evolving second internal transcribed spacer (ITS-2). The whole 18S rRNA gene is a useful marker for supra-specific relationships in Triatominae. ITS-2 is complementary to it, enabling resolution at specific and infraspecific levels. All the evidence suggests that ITS-2 will become the DNA marker of excellence for studies of Triatominae at specific and subspecific levels, as it is in other groups of organisms. Possible applications of data obtained from the study of rRNA and ITS-2 sequences of Triatominae are discussed.

Genomic structure, chromosome mapping and expression analysis of the human AVIL gene, and its exclusion as a candidate for locus for inflammatory bowel disease at 12q13–14 (IBD2)

Chronic inflammatory bowel disease is a multifactorial disorder with two major clinical forms, Crohn's disease and ulcerative colitis. One of the potential susceptibility loci for inflammatory bowel disease (IBD2) was localized at 12q13–14 in the vicinity of the deoxyribonucleic acid marker D12S83 by linkage analysis. A candidate susceptibility gene for IBD2 in this region is the AVIL gene. AVIL encodes a protein (advillin) which belongs to the gelsolin/villin family of proteins and might therefore be involved in morphogenesis of microvilli. We have determined the genomic organization of the AVIL gene, including the transcription start site and its localization with respect to D12S83. The 2457 bp coding region of AVIL consists of 19 exons and is localized to 12q14 proximal to D12S83. Primer extension analysis suggests two transcription start sites localized at −548 and −664 bp upstream to the ATG translation codon. We have evaluated AVIL as a candidate susceptibility gene for IBD2 in 24 unrelated patients with evidence of linkage to chromosome 12, as well as in 91 individuals from 19 affected IBD families for putative single nucleotide polymorphisms.

Detection of maternal deoxyribonucleic acid in umbilical cord plasma by using fluorescent polymerase chain reaction amplification of short tandem repeat sequences

Objective: Umbilical cord blood is a source of hematopoietic stem cells for transplantation. Although the first clinical applications have been encouraging, concern has been raised about contamination of umbilical blood by maternal cells, which might constitute a theoretical risk of graft-versus-host disease. The aim of this study was to assess the frequency of maternal deoxyribonucleic acid (DNA) contamination in umbilical cord plasma by using fluorescent polymerase chain reaction amplification of highly polymorphic short tandem repeat DNA markers. Study Design: Fifty-seven mother/child pairs were tested for the presence of maternal DNA sequences in cord plasma. After delivery, cord blood samples were collected via gravity. Maternal specific alleles were detected by using polymerase chain reaction amplification of 9 highly polymorphic short tandem repeat markers (D21S11, D21S1411, D21S1412, D18S386, D18S535, MBP-A, MBP-B, D13S631, and D13S634). Results: All 57 mother-child pairs were informative for the identification of uniquely maternal alleles in at least 2 of 9 different short tandem repeat markers used per case. Uniquely maternal DNA sequences were found in 43 of 57 (75%) cord plasma samples. Conclusion: The results of our study demonstrate that maternal DNA is present in the majority of umbilical cord blood plasma samples. The technique described herein might have application in the screening of umbilical cord blood samples for the presence of contaminating maternal genetic material. (Am J Obstet Gynecol 2002;186:117-20.)

Microsatellite alterations in exfoliated cervical epithelia deoxyribonucleic acid as a marker for high-grade dysplasia

Objective: The purpose of this study was to evaluate the feasibility of the use of deoxyribonucleic acid microsatellite alterations in cervical epithelia in the prediction of high-grade dysplasia and to compare it with a strategy based on human papillomavirus testing. Study Design: Our prospective study subjects were from a cohort of 498 women with minimally abnormal Papanicolaou test results including atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion who had documented repeated Papanicolaou and human papillomavirus tests. Of these, 52 eligible patients having conizations or hysterectomies as their histologic outcomes were subjected to tests of loss of heterozygosity on a panel of 5 microsatellites (D3S1110, THRB, D3S1228, D6S291, D3S1289) within the deoxyribonucleic acid of exfoliated cervical epithelia. These genetic alterations were analyzed through fluorescence polymerase chain reaction by comparison of allele ratios of exfoliated cells with those of normal control tissue. Predictive values for high-grade cervical dysplasia and cancer between this deoxyribonucleic acid marker and human papillomavirus status were compared. Results: With the use of loss of heterozygosity in at least one locus for predicting high-grade cervical neoplastic lesion, the sensitivity, specificity, positive predictive value, and negative predictive value were 96.7%, 59.1%, 76.3%, and 92.9%, which were superior to those of the human papillomavirus test (80%, 59.1%, 72.7%, and 92.9%). As a triage for atypical squamous cells of undetermined significance, its sensitivity and negative predictive value were up to 100%. Conclusion: The promising results on determining microsatellite alteration in dysplastic lesions might imply that it is possible to detect the earliest changes by potential molecular markers with exfoliated cervical epithelial cells. (Am J Obstet Gynecol 2001;185:108-15.)

Genetic Exchange in the Trypanosomatidae

The only trypanosomatid so far proved to undergo genetic exchange is Trypanosoma brucei, for which hybrid production after co-transmission of different parental strains through the tsetse fly vector has been demonstrated experimentally. Analogous mating experiments have been attempted with other Trypanosoma and Leishmania species, so far without success. However, natural Leishmania hybrids, with a combination of the molecular characters of two sympatric species, have been described amongst both New and Old World isolates. Typical homozygotic and heterozygotic banding patterns for isoenzyme and deoxyribonucleic acid markers have also been demonstrated amongst naturally-occurring T. cruzi isolates. The mechanism of genetic exchange in T. brucei remains unclear, although it appears to be a true sexual process involving meiosis. However, no haploid stage has been observed, and intermediates in the process are still a matter for conjecture. The frequency of sex in trypanosomes in nature is also a matter for speculation and controversy, with conflicting results arising from population genetics analysis. Experimental findings for T. brucei are discussed in the first section of this review, together with laboratory evidence of genetic exchange in other species. The second section covers population genetics analysis of the large body of data from field isolates of Leishmania and Trypanosoma species. The final discussion attempts to put the evidence from experimental and population genetics into its biological context.

The incidence of uniparental disomy associated with intrauterine growth retardation in a cohort of thirty-five severely affected babies

OBJECTIVE: Our purpose was to screen for uniparental disomy 35 babies with idiopathic intrauterine growth retardation <5th percentile. STUDY DESIGN: The placenta and the baby's blood were conventionally karyotyped. Deoxyribonucleic acid from the parents, the baby's blood, and the placenta were then screened for uniparental disomy for 12 candidate chromosomes with use of chromosome-specific polymorphic deoxyribonucleic acid markers. RESULTS: Two cases of maternal uniparental disomy for chromosome 16 were found associated with confined placental mosaicism for chromosome 16. No other uniparental disomy was found for any of the 12 chromosomes tested. Four structural chromosome abnormalities were also found in this cohort through standard karyotyping. CONCLUSION: Uniparental disomy for the chromosomes tested does not explain the etiology of the majority of cases of intrauterine growth retardation <5th percentile. Maternal uniparental disomy for chromosome 16 accounts for 5% of this cohort. Structural chromosomal abnormalities are also much higher than expected at 11%.(Am J Obstet Gynecol 1997;176:294-9.)

Nicotinic receptor function in schizophrenia.

Schizophrenia can be partially characterized by deficits in sensory processing. Biochemical, molecular, and genetic studies of one such endophenotype, the P50 auditory-evoked potential gating deficit, suggest that one of the neuronal nicotinic receptors, the alpha 7 nicotinic receptor, may function in an inhibitory neuronal pathway involved in this phenotype. The P50 deficit is normalized in nongating subjects by nicotine. Although most schizophrenia patients are heavy smokers, the effects of nicotine may be transient, as alpha 7 receptors are known to desensitize rapidly. In an animal model of the P50 gating deficit, antagonists of the alpha 7 nicotinic receptor block normal gating of the second of paired auditory stimuli. Regional localization of receptor expression includes areas known to function in sensory filtering. An inhibitory mechanism, in the hippocampus, may involve nicotinic stimulation of gamma-aminobutyric acid (GABA)ergic interneurons, resulting in decreased response to repetitive stimuli. Expression of the alpha 7 receptor is decreased in hippocampal brain tissue, dissected postmortem, from schizophrenia subjects. The P50 deficit is inherited in schizophrenia pedigrees, but it is not sufficient for disease development and thus represents a predisposition factor. Linkage analysis between the P50 deficit in multiplex schizophrenia pedigrees and deoxyribonucleic acid (DNA) markers throughout the genome yielded positive lod scores to DNA markers mapping to a region of chromosome 15 containing the alpha 7 nicotinic receptor gene. Elucidation of possible interactions of the P50 with other factors, known to be important in the etiology of the disease, is important in determining an overall pathobiology of schizophrenia.

Genetic Basis of Familial Dyslipidemia and Hypertension: 15-Year Results From Utah

The genetic and environmental determinants of hypertension, lipid abnormalities, and coronary artery disease (CAD) have been studied for 15 years in Utah in population-based multigenerational pedigrees (2500 subjects among 98 pedigrees), twin pairs (74 monozygous and 78 dizygous), hypertensive siblings (131 sibships), siblings with CAD before age 55 (45 sibships), and anecdotally ascertained pedigrees with type II diabetes (271 subjects among 16 pedigrees), lipoprotein lipase deficiency (106 subjects in a single pedigree), and familial hypercholesterolemia (502 heterozygotes among 50 pedigrees). Estimates of heritability ranged from 20 to 75% for blood pressures and blood lipids. A strong positive family history predicts a future occurrence of hypertension (relative risk [RR] = 3.8) and CAD (RR = 12.7). Segregating single-gene effects were found for several 'intermediate phenotypes' associated with hypertension (erythrocyte sodium-lithium countertransport, intraerythrocytic sodium, a relative fat pattern, total urinary kallikrein excretion, and fasting insulin levels). Strong single-gene effects in segregation analysis were also found for low-density lipoprotein (LDL) cholesterol, lipoprotein (a) (Lp[a]), low high-density lipoprotein (HDL) cholesterol, and high apolipoprotein (apo) B. Deoxyribonucleic acid (DNA) markers of lipid abnormalities or hypertension have included LDL-receptor defects, lipoprotein lipase deficiency, high Lp(a), familial defective apo B, decreased quantitative levels of apo B, apo E phenotype, angiotensinogen, and 'glucocorticoid remediable aldosteronism (GRA) hypertension.' Also tested in Utah studies, but not found to be DNA markers for hypertension, were the genetic loci for the structural genes for renin and angiotensin-converting enzyme, and the sodium antiport system. In addition, important gene-gene interactions (LDL receptor with apo E2) and gene-environment interactions (kallikrein with potassium intake) were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone-secreting adrenal adenoma as part of multiple endocrine neoplasia type 1 (MEN1): loss of heterozygosity for polymorphic chromosome 11 deoxyribonucleic acid markers, including the MEN1 locus

Aldosterone-secreting adrenal adenoma as part of multiple endocrine neoplasia type 1 (MEN1): loss of heterozygosity for polymorphic chromosome 11 deoxyribonucleic acid markers, including the MEN1 locus

Prenatal diagnosis of myotonic muscular dystrophy with linked deoxyribonucleic acid probes

Since the localization of the myotonic muscular dystrophy gene, closer deoxyribonucleic acid markers have been discovered. These now facilitate both presymptomatic and prenatal diagnosis of myotonic muscular dystrophy. We report our prenatal diagnosis experience with six cases in five families. Obstetricians are advised to inform their patients with a family history of myotonic muscular dystrophy of these testing opportunities. The fetus of the mother with myotonic muscular dystrophy who remains in utero until term is at considerable risk, as is the mother herself, of serious obstetric complications.

Molecular genetics of inherited antithrombin III deficiencies

The cloning of antithrombin III (ATIII) complementary deoxyribonucleic acids and the determination of the ATIII gene structure have permitted a systematic evaluation of the molecular basis for inherited ATIII deficiencies. Sixteen kindreds with the most common form of the deficiency, in which plasma ATIII antigen levels and activity are proportionately reduced, were studied. Two polymorphic deoxyribonucleic acid markers were used to resolve parental ATIII alleles and to trace their inheritance patterns. In 15 of 16 cases, the structure of the affected ATIII allele was indistinguishable from normal, suggesting that relatively small mutations, resulting in gene inactivation, are responsible for the lower ATIII levels in these affected families. In the remaining kindred, complete deletion of one ATIII allele was seen. Also investigated was the molecular basis for a qualitative form of ATIII deficiency in a French-Canadian family with normal levels of immunoreactive protein but only half the expected levels of serine protease inhibitor activity. Using polymorphic markers, the abnormal allele was identified, cloned, and partially sequenced from the propositus. A single G----A transition was seen in the first base of codon 382, resulting in an alanine----threonine substitution in the defective protein. This mutation, together with others in this vicinity, defines a minimal length for a fully functional thrombin-binding domain.

Clinical application of deoxyribonucleic acid markers in a Utah family with hypercholesterolemia

The manifestation of coronary artery disease (CAD) depends on the interplay of genetic and environmental factors. 1 Deoxyribonucleic acid (DNA) markers should become practical clinical tools for examining the genetic components of CAD. With this in mind, we assessed the use of DNA markers in familial hypercholesterolemia. Many different DNA mutations of the low density lipoprotein receptor gene on chromosome 19 cause familial hypercholesterolemia. 2 Familial hypercholesterolemia is a prevalent autosomal trait characterized by elevated levels of plasma low density lipoprotein cholesterol and early CAD. Heterozygotes have serum cholesterol levels >300 mg/dl and affected men often develop CAD by the fifth decade of life. 2 Restriction fragment length polymorphisms are DNA variations that are detected by Southern blotting. 3 DNA differences between people show up as different sized allelic bands on autoradiograms of Southern blots. Investigators can follow a gene's inheritance by studying the pattern of allelic bands within families of interest. 3 If a DNA marker appears consistently with the disease in affected family members, then DNA variation at the marker locus is said to be linked with the disease. 3 Once linkage with a genetic locus is established, secondary influences on the disease such as diet or exercise can be examined. In this study, we report an 8-generation Utah kindred with many members affected with familial hypercholesterolemia. We found that the familial hypercholesterolemia phenotype was due to a mutation at the low density lipoprotein receptor gene locus. A wide discrepancy in the age of coronary deaths among gene carriers suggested the influence of other genetic or environmental factors. The DNA markers also helped to diagnose as normal a child with borderline high cholesterol levels clinically suspected to have familial hypercholesterolemia.

Impact of molecular biology on clinical genetics.

The central activity of clinical genetics is genetic counselling, the aim of which is to provide information about the risks to offspring at a time appropriate to considering the options available for modifying the outcome and to put those risks in perspective. There is much evidence to show that with serious or life threatening disorders genetic counselling causes many couples to modify their reproductive behaviour, even when the options available are far from satisfactory. Our recently acquired ability to use deoxyribonucleic acid (DNA) probes (radiolabelled single stranded DNA fragments that bind only to specific genes or DNA sequences from the subject's total DNA) to analyse mutations or track the inheritance of genes through families has enormous potential as an aid to genetic counselling. Couple this with the development of chorionic villus sampling in the first trimester of pregnancy and the effect for some families is nothing short of revolutionary. DNA analysis was first used in the prenatal diagnosis of the haemoglobinopathies. Kan and Dozy reported the diagnosis of sickle cell anaemia on amniotic cells taken in the second trimester,1 and Old et al were first to report fetal diagnosis of ? thalassaemia in the first trimester based on the analysis of chorionic villus DNA.2 Since then so many monogenic disorders have become amenable to this approach that it is not possible to give a comprehensive up to date list. Schmidtke and Cooper provide a regular listing of cloned human genes or DNA sequences that can be used as DNA markers.3 Their list of independently cloned DNA sequences published in April 1987 contains nearly 3000 entries. The table gives those common conditions in which prenatal or carrier tests are already being used and which constitute most of the activity of genetics units providing DNA analysis as a clinical service. There is no correlation between the incidence of a disorder and the chances that the relevant gene has been mapped or even cloned. No matter how rare the genetic disease specific inquiries on behalf of a family at risk should always be made, especially in X linked disorders. The interval between gene localisation and clinical application of this information can be very short. Within months of the cystic fibrosis mutation being mapped to the 7q22-31 region of chromosome 7 some lucky couples who had not risked a pregnancy since the diagnosis was made in their child were having prenatal diagnosis in the first trimester.41 say lucky couples because gene tracking (see below) with DNA probes usually helps only certain families. These couples were also lucky because the service was offered to them. The National Health Service is not

The use of flanking markers in prediction for Duchenne muscular dystrophy.

Seventy three sisters of boys with Duchenne muscular dystrophy and their families were studied using up to seven deoxyribonucleic acid (DNA) probes linked to the gene on the short arm of the X chromosome. Fifty three (73%) were informative for flanking markers, a further 18 (24%) being informative for a single marker only. Predictions based on pedigree structure and creatine kinase information from the individuals and their female relatives gave more than half (55%) of the sisters a risk for being a carrier of below 10% or above 90%. The addition of information derived from the inheritance of flanking DNA markers, where they exist, improves the situation so that many more sisters (77%) can be allocated into either high or low risk categories.

First trimester prenatal diagnosis and detection of carriers of haemophilia A using the linked DNA probe DX13.

Although the use of a gene specific deoxyribonucleic acid (DNA) probe is the method of choice for detecting carriers of genes for rare genetic disorders, there will always be families in which such probes cannot be used because key subjects are not informative for restriction fragment length polymorphisms in or around the gene. In these cases closely linked DNA markers have to be used. An X chromosome specific DNA probe, DX13, which is closely linked to the haemophilia A locus on the X chromosome, was used for early prenatal diagnosis in two cases and to detect carriers in a series of nine possible heterozygote women. The first reported crossover between DX13 and the factor VIII:C locus was observed in this study. There are complexities inherent in using any linked DNA probe for assignment of genes, but such techniques are clinically important.

Genetic prediction and family structure in Huntington's chorea.

A deoxyribonucleic acid marker linked to the locus for Huntington's chorea exists, but its possible use in the prediction of this disorder depends on the pedigree structure of individual families. Analysis of data from a population register for Huntington's chorea in south Wales showed that only a minority of subjects at risk had the appropriate members of their family living to allow the presence or absence of the gene to be definitively predicted. However, the structure of the family allowed a degree of prediction (in particular, exclusion of the disorder) to be made for the fetus during pregnancies of these subjects in almost 90% of cases. Such a prediction need not alter the risk state for the parent at risk. The structure of the family will remain crucial for prediction even when current limitations of the linked marker have been overcome.

Fate of transforming deoxyribonucleic acid after uptake by competent Bacillus subtilis: nonrequirement of deoxyribonucleic acid replication for uptake and integration of transforming deoxyribonucleic acid.

Studies on transformation of Bacillus subtilis using the inhibitor 6-(p-hydroxyphenylazo)-uracil show that deoxyribonucleic acid (DNA) replication is not required for the uptake and integration of donor DNA and genetic markers.

Recovery of donor deoxyribonucleic acid marker activity from eclipse in pneumococcal transformation.

After the uptake of deoxyribonucleic acid (DNA), donor marker-transforming activity is temporarily lost. Restoration of the activity by annealing in vitro supports the idea that donor DNA is single-stranded at this stage. Kinetics of in vivo recovery from eclipse were examined for various markers at three temperatures. Sigmoidal recovery curves at lower temperatures indicate that the process consists of several steps. Rate of recovery was found to depend on the nature of the donor marker. Single-site markers recover much more rapidly than multisite markers corresponding to recipient deletions. Single-site markers vary somewhat in recovery rate, with rapidity of recovery inversely related to integration efficiency. Appearance of a recombinant-transforming activity lags only slightly behind recovery of its constituent donor marker.