Medical genetics was revolutionized during the 1980s by the application of genetic mapping to locate the genes responsible for simple Mendelian diseases. Most diseases and traits, however, do not follow simple inheritance patterns. Geneticists have thus begun taking up the even greater challenge of the genetic dissection of complex traits. Four major approaches have been developed: linkage analysis, allele-sharing methods, association studies, and polygenic analysis of experimental crosses. This article synthesizes the current state of the genetic dissection of complex traits—describing the methods, limitations, and recent applications to biological problems.

Genetic Dissection of Complex Traits

http://hemepathreview.com/UsefulHemeRefArticles/Monitor-CML-Standards.pdf

Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results

Severe myoclonic epilepsy of infancy (SMEI) is a rare disorder that occurs in isolated patients. The disease is characterized by generalized tonic, clonic, and tonic-clonic seizures that are initially induced by fever and begin during the first year of life. Later, patients also manifest other seizure types, including absence, myoclonic, andsimple and complex partial seizures. Psychomotor development stagnates around the second year of life. Missense mutations in the gene that codes for a neuronal voltage-gated sodium-channel α-subunit (SCN1A) were identified in families with generalized epilepsy with febrile seizures plus (GEFS+). GEFS+ is a mild type of epilepsy associated with febrile and afebrile seizures. Because both GEFS+ and SMEI involve fever-associated seizures, we screened seven unrelated patients with SMEI for mutations in SCN1A. We identified a mutation in each patient: four had frameshift mutations, one had a nonsense mutation, one had a splice-donor mutation, and one had a missense mutation. All mutations are de novo mutations and were not observed in 184 control chromosomes.

De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy.

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.

/pdf/accessing-genetic-variation-genotyping-single-nucleotide-5a62oxngy2.pdf

Accessing genetic variation: genotyping single nucleotide polymorphisms

DNA sequencing is one of the most important platforms for the study of biological systems today. Sequence determination is most commonly performed using dideoxy chain termination technology. Recently, pyrosequencing has emerged as a new sequencing methodology. This technique is a widely applicable, alternative technology for the detailed characterization of nucleic acids. Pyrosequencing has the potential advantages of accuracy, flexibility, parallel processing, and can be easily automated. Furthermore, the technique dispenses with the need for labeled primers, labeled nucleotides, and gel-electrophoresis. This article considers key features regarding different aspects of pyrosequencing technology, including the general principles, enzyme properties, sequencing modes, instrumentation, and potential applications.

/pdf/pyrosequencing-sheds-light-on-dna-sequencing-4vpt46skyo.pdf

Pyrosequencing sheds light on DNA sequencing.

The characterization of naturally occurring variations in the human genome has evoked an immense interest during recent years. Variations known as biallelic Single-Nucleotide Polymorphisms (SNPs) have become increasingly popular markers in molecular genetics because of their wide application both in evolutionary relationship studies and in the identification of susceptibility to common diseases. We have addressed the issue of SNP genotype determination by investigating variations within the Renin-Angiotensin-Aldosterone System (RAAS) using pyrosequencing, a real-time pyrophosphate detection technology. The method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. The technical platform employed comprises a highly automated sequencing instrument that allows the analysis of 96 samples within 10 to 20 minutes. In addition to each studied polymorphic position, 5-10 downstream bases were sequenced for acquisition of reference signals. Evaluation of pyrogram data was accomplished by comparison of peak heights, which are proportional to the number of incorporated nucleotides. Analysis of the pyrograms that resulted from alternate allelic configurations for each addressed SNP revealed a highly discriminating pattern. Homozygous samples produced clear-cut single base peaks in the expected position, whereas heterozygous counterparts were characterized by distinct half-height peaks representing both allelic positions. Whenever any of the allelic bases of an SNP formed a homopolymer with adjacent bases, the nonallelic signal was added to those of the SNP. This feature did not, however, influence SNP readability. Furthermore, the multibase reading capacity of the described system provides extensive flexibility in regard to the positioning of sequencing primers and allows the determination of several closely located SNPs in a single run.

/pdf/determination-of-single-nucleotide-polymorphisms-by-real-3lqwzohkon.pdf

Determination of Single-Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing

With a large part of the Human Genome Project behind us and several smaller genomes already finished, we are facing new challenges. Until now most of the collective effort has been focused on sequencing genomes, but the activities to interpret and use this information are now rapidly accelerating. Technology developed for the long read lengths typically required for sequencing projects are not necessarily well suited for the emerging needs of applied genomics where identification of known genes or the study of Single Nucleotide Polymorphism, can be achieved with only a few bases of sequence information. Several new methods such as Taqman, Molecular Beacons, Invader Probes, Mini sequencing (single base extension) etc are being developed to meet these new needs but very few of these has the ability to read more than one base. This paper describes Pyrosequencing, a new way of sequencing DNA based on Sequencing By Synthesis and with the ability to sequence short to medium length stretches of DNA with high accuracy.

Ulf Hammerling

Papers

Determination of Single-Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing

Pyrosequencing for SNPs