Understanding the relationship between genetic variation and biological function on a genomic scale is expected to provide fundamental new insights into the biology, evolution and pathophysiology of humans and other species. The hope that single nucleotide polymorphisms (SNPs) will allow genes that underlie complex disease to be identified, together with progress in identifying large sets of SNPs, are the driving forces behind intense efforts to establish the technology for large-scale analysis of SNPs. New genotyping methods that are high throughput, accurate and cheap are urgently needed for gaining full access to the abundant genetic variation of organisms.

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Accessing genetic variation: genotyping single nucleotide polymorphisms

During the last ten years, the use of molecular markers, revealing polymorphism at the DNA level, has been playing an increasing part in animal genetics studies. Amongst others, the microsatellite DNA marker has been the most widely used, due to its easy use by simple PCR, followed by a denaturing gel electrophoresis for allele size determination, and to the high degree of information provided by its large number of alleles per locus. Despite this, a new marker type, named SNP, for Single Nucleotide Polymorphism, is now on the scene and has gained high popularity, even though it is only a bi-allelic type of marker. In this review, we will discuss the reasons for this apparent step backwards, and the pertinence of the use of SNPs in animal genetics, in comparison with other marker types.

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A review on SNP and other types of molecular markers and their use in animal genetics

Editor—Anderson-Fabry disease (AFD) is a sphingolipid storage disorder resulting from the deficiency of the lysosomal enzyme α-galactosidase. Unlike most other lysosomal diseases, the inheritance is X linked. Disease manifestations in female heterozygotes have been reported, but are considered to be rare and usually mild.1 Asymptomatic corneal dystrophy (cornea verticillata and posterior lenticular cataract) is present in about 70% and is useful for heterozygote detection. About 30% of women have minimal angiokeratomas and <10% have infrequent attacks of neuropathic pains.1 However, female heterozygotes with severe and early cerebrovascular disease, strokes, and renal failure have been documented, but these serious manifestations were estimated to occur in only 1%.1 Because this is an X linked disorder, these severe manifestations in females were explained by skewed X inactivation.2 

Case reports are open to selection bias and there are no reported data on the frequency and severity of AFD manifestations in a large cohort of carriers. Women are not usually evaluated in the clinic unless they present with serious AFD complications, but they do attend for genetic counselling when their son or male relative is diagnosed with the disease.

During regular reviews of AFD families in the genetic clinic, we have observed multiple and more frequent AFD manifestations in female carriers than expected. Owing to the lack of clinical data and present health care policy, we were unable to perform clinical investigations. Therefore, a large cohort study using an AFD specific questionnaire was performed to establish the mortality and the frequency of all AFD manifestations in obligate carrier females.

All known (n=80) obligate carrier females, who were over the age of 18 and had demographic data recorded in the AFD register, were asked to participate in this study. Sixty (75%) agreed and provided data; non-responders were reminded on one …

Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 60 obligate carrier females

The present invention relates to the detection of nucleic acid sequence differences using coupled ligase detection reaction and polymerase chain reaction. One aspect of the present invention involves use of a ligase detection reaction coupled to a polymerase chain reaction. Another aspect of the present invention relates to the use of a primary polymerase chain reaction coupled to a secondary polymerase chain reaction coupled to a ligase detection reaction. A third aspect of the present invention involves a primary polymerase chain reaction coupled to a secondary polymerase chain reaction. Such coupling of the ligase detection reaction and the polymerase chain reaction permits multiplex detection of nucleic acid sequence differences.

Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions

Synthetic oligonucleotide analogs have greatly aided our understanding of several biochemical processes. Efficient solid-phase and enzyme-assisted synthetic methods and the availability of modified base analogs have added to the utility of such oligonucleotides. In this review, we discuss the applications of synthetic oligonucleotides that contain backbone, base, and sugar modifications to investigate the mechanism and stereochemical aspects of biochemical reactions. We also discuss interference mapping of nucleic acid-protein interactions; spectroscopic analysis of biochemical reactions and nucleic acid structures; and nucleic acid cross-linking studies. The automation of oligonucleotide synthesis, the development of versatile phosphoramidite reagents, and efficient scale-up have expanded the application of modified oligonucleotides to diverse areas of fundamental and applied biological research. Numerous reports have covered oligonucleotides for which modifications have been made of the phosphodiester backbone, of the purine and pyrimidine heterocyclic bases, and of the sugar moiety; these modifications serve as structural and mechanistic probes. In this chapter, we review the range, scope, and practical utility of such chemically modified oligonucleotides. Because of space limitations, we discuss only those oligonucleotides that contain phosphate and phosphate analogs as internucleotidic linkages.

MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users

We describe a non-isotopic, semi-automated method for large-scale multiplex analysis of nucleic acid sequences, using the cystic fibrosis transmembrane regulator (CFTR) gene as an example. Products of a multiplex oligonucleotide ligation assay (OLA) are resolved electrophoretically from one another and from unligated probes under denaturing conditions with fluorescence detection. One ligation probe for each OLA target carries a fluorescent tag, while the other probe carries an oligomeric non-nucleotide mobility modifier. Each OLA product has a unique electrophoretic mobility determined by the ligated oligonucleotides and the mobility-modifier oligomer arbitrarily assigned (coded) to its target. The mobility range for practical mobility modifiers is much wider than the accessible range from unmodified ligated oligonucleotides of practical length. Each mobility modifier is built from phosphoramidite monomers in a stepwise manner on its associated oligonucleotide using an automated synthesizer. The resulting mobility modifiers lower the probe-target duplex Tm by less than 3 degrees C and retard probe-target annealing by less than 50%, with negligible effect on OLA yield and specificity. This method is especially useful for allelic discrimination in highly polymorphic genes such as CFTR.

High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation.

A method based on polymerase chain reaction (PCR) amplification and oligonucleotide ligase assay (OLA) reaction is provided for analyzing complex genetic systems in a single reaction vessel The method involves simultaneously incubating a sample containing one or more target polynucleotides with PCR primers and OLA probes in a single reaction mixture The presence of variant polynucleotide sequences in the sample is determined by detecting and identifying the products of the OLA reaction

Coupled amplification and ligation method

Isolation of the gene for cystic fibrosis (CF), the cystic fibrosis transmembrane conductance regulator (CFTR), provided a basis for analyzing its molecular pathology and resulted in the identification of > 400 mutations associated with disease. Except for the delta F508 mutation, no other single mutation accounts for > 5% of CF chromosomes in most populations, and most mutation frequencies are 96% of CF mutant chromosomes worldwide. Mutations were detected by competitive oligonucleotide probe ligation to detect normal and/or mutant genotypes in one reaction. Three probes (one common and two allelic probes) were needed for analysis of each mutation. Probes hybridized to target DNA were joined by a thermostable ligase if there were no mismatches at their junctions; temperature cycling resulted in a linear increase in product. Common probes were labeled with fluorochromes, and allelic probes each had different lengths. Ligation products were analyzed electrophoretically on a fluorescent DNA sequencer. The results show that combined PCR and probe ligation amplification rapidly and reliably screen for CF homozygotes and carriers.

Fluorescence-based oligonucleotide ligation assay for analysis of cystic fibrosis transmembrane conductance regulator gene mutations

A new technique, coupled amplification and oligonucleotide ligation (CAL), has been developed that allows for simultaneous multiplex amplification and genotyping of DNA. CAL is a biphasic method that combines in one assay DNA amplification by PCR with DNA genotyping by the oligonucleotide ligation assay (OLA). By virtue of a difference in the melting temperatures of PCR primer-target DNA and OLA probe-target DNA hybrids, the method allows preferential amplification of DNA during stage I and oligonucleotide ligation during stage II of the reaction. In stage I, target DNA is amplified using high-melting primers (Tm values between 68 degrees C and 89 degrees C) in a two-step PCR cycle that employs a 94 degrees C denaturation step and a 72 degrees C anneal-elongation step. In stage II, genotyping of PCR products by competitive oligonucleotide ligation with oligonucleotide probes (Tm values between 51 degrees C and 67 degrees C) located between the PCR primers is accomplished by several cycles of denaturation at 94 degrees C followed by anneal-ligation at 55 degrees C. Ligation products are fluorochrome-labeled at their 3' ends and analyzed electrophoretically on a fluorescent DNA sequencer. The CAL procedure has been used successfully to analyze human genomic DNA for cystic fibrosis (CF) alleles. Because product detection occurs concurrently with target amplification, the technique is rapid, highly sensitive, and specific and requires minimal sample processing.

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A one-step coupled amplification and oligonucleotide ligation procedure for multiplex genetic typing

The field of medical, molecular diagnostics has grown rapidly over the last few years, becoming increasingly informative to both clinician and patient. As genes associated with specific diseases have been discovered and sequenced, many genotype-phenotype relationships have been defined. For those genetic diseases with associated, defined, gene mutations, sophisticated DNA diagnostic tests are being developed. As an example, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, is associated with Cystic Fibrosis (CF). We have developed a new molecular diagnostic technology, PCR/OLA/SCS, and applied it first to the diagnosis of CF. Test design in the field of molecular diagnostics must consider such characteristics as specificity, sensitivity, ease and speed of protocol, multiplex capacity, and cost. PCR/OLA/SCS addresses these requirements. Polymerase Chain Reaction (PCR) is widely used in both diagnostics and research. We have combined well established PCR technology with Oligonucl...

Faye A. Eggerding

Papers

High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation.

Coupled amplification and ligation method

Fluorescence-based oligonucleotide ligation assay for analysis of cystic fibrosis transmembrane conductance regulator gene mutations

A one-step coupled amplification and oligonucleotide ligation procedure for multiplex genetic typing

Introduction to PCR/OLA/SCS, a multiplex DNA test, and its application to cystic fibrosis