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

The real-time polymerase chain reaction (PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction. This combines the DNA amplification and detection steps into one homogeneous assay and obviates the need for gel electrophoresis to detect amplification products. Appropriate data analysis and/or use of apposite chemistries also eliminates the need for Southern blotting or DNA sequencing for amplicon identification. Its simplicity, specificity, and sensitivity, together with its potential for high throughput and the ongoing introduction of new chemistries, more reliable instrumentation, and improved protocols, has made realtime PCR the benchmark technology for the detection of DNA.

Real-Time PCR

Marker-assisted selection (MAS) refers to the use of molecular markers to assist phenotypic selections in crop improvement. Several types of molecular markers, such as single nucleotide polymorphism (SNP), have been identified and effectively used in plant breeding. The application of next-generation sequencing (NGS) technologies has led to remarkable advances in whole genome sequencing, which provides ultra-throughput sequences to revolutionize plant genotyping and breeding. To further broaden NGS usages to large crop genomes such as maize and wheat, genotyping-by-sequencing (GBS) has been developed and applied in sequencing multiplexed samples that combine molecular marker discovery and genotyping. GBS is a novel application of NGS protocols for discovering and genotyping SNPs in crop genomes and populations. The GBS approach includes the digestion of genomic DNA with restriction enzymes followed by the ligation of barcode adapter, PCR amplification and sequencing of the amplified DNA pool on a single lane of flow cells. Bioinformatic pipelines are needed to analyze and interpret GBS datasets. As an ultimate MAS tool and a cost-effective technique, GBS has been successfully used in implementing genome-wide association study (GWAS), genomic diversity study, genetic linkage analysis, molecular marker discovery and genomic selection under a large scale of plant breeding programs.

/pdf/genotyping-by-sequencing-gbs-an-ultimate-marker-assisted-2qv3na21mn.pdf

Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding.

We have developed a new class of probes for homogeneous nucleic acid detection based on the proposed displacement hybridization. Our probes consist of two complementary oligodeoxyribonucleotides of different length labeled with a fluorophore and a quencher in close proximity in the duplex. The probes on their own are quenched, but they become fluorescent upon displacement hybridization with the target. These probes display complete discrimination between a perfectly matched target and single nucleotide mismatch targets. A comparison of double-stranded probes with corresponding linear probes confirms that the presence of the complementary strand significantly enhances their specificity. Using four such probes labeled with different color fluorophores, each designed to recognize a different target, we have demonstrated that multiple targets can be distinguished in the same solution, even if they differ from one another by as little as a single nucleotide. Double-stranded probes were used in real-time nucleic acid amplifications as either probes or as primers. In addition to its extreme specificity and flexibility, the new class of probes is simple to design and synthesize, has low cost and high sensitivity and is accessible to a wide range of labels. This class of probes should find applications in a variety of areas wherever high specificity of nucleic acid hybridization is relevant.

/pdf/a-new-class-of-homogeneous-nucleic-acid-probes-based-on-19dfptfwu0.pdf

A new class of homogeneous nucleic acid probes based on specific displacement hybridization

Current serologic tests provide the foundation for diagnosis of hepatitis A and hepatitis A virus (HAV) infection. Recent advances in methods to identify and characterize nucleic acid markers of viral infections have provided the foundation for the field of molecular epidemiology and increased our knowledge of the molecular biology and epidemiology of HAV. Although HAV is primarily shed in feces, there is a strong viremic phase during infection which has allowed easy access to virus isolates and the use of molecular markers to determine their genetic relatedness. Molecular epidemiologic studies have provided new information on the types and extent of HAV infection and transmission in the United States. In addition, these new diagnostic methods have provided tools for the rapid detection of food-borne HAV transmission and identification of the potential source of the food contamination.

Diagnosis of Hepatitis A Virus Infection: a Molecular Approach

Homogeneous assays based on real-time fluorescence monitoring during PCR are relevant alternatives for large-scale genotyping of single-nucleotide polymorphisms (SNPs). We compared the performance ...

https://www.future-science.com/doi/pdf/10.2144/00284rr02

M. Wik

Papers

Homogeneous Scoring of Single-Nucleotide Polymorphisms: Comparison of the 5′-Nuclease TaqMan® Assay and Molecular Beacon Probes