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FISH - Fluorescent In-situ Hybridization

Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in cytogenetics. Labeled probes are used to bind complementary DNA or RNA sequences on a chromosome or in a region within a cell. Earlier, the probes could only be obtained by cloning or reverse transcription of a DNA template. Currently, the probe oligonucleotides can be synthesized synthetically. Additionally, with the advancement of optical techniques,...
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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations
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Fluorescence-Based Absent Allele-Specific Amplification (FAASA) for High-Throughput Detection of Absent Alleles.

Katherine L D Running1, Sudeshi Seneviratne1, Zengcui Zhang2

  • 1Department of Plant Sciences, North Dakota State University, Fargo, ND, USA.

Bio-Protocol
|April 2, 2026
PubMed
Summary

A new Fluorescence-based Absent Allele-Specific Amplification (FAASA) method reliably detects gene absence in crops. This robust genotyping technique overcomes limitations of existing methods, enabling precise marker-assisted selection and genetic studies.

Keywords:
GenotypingKASPMarker-assisted selectionPACEPresence/absence variationWheat

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Area of Science:

  • Plant genetics and breeding
  • Molecular biology
  • Genotyping technologies

Background:

  • Presence/absence variation in crop genes is important for trait selection, but detecting absent alleles is challenging.
  • Existing high-throughput genotyping methods like KASP and PACE struggle with absent alleles, as failed PCR is indistinguishable from gene absence.
  • Accurate detection of gene absence is crucial for marker-assisted selection and genetic analysis in crops.

Purpose of the Study:

  • To develop a reliable and high-throughput method for detecting the absence of specific gene alleles in crops.
  • To overcome the limitations of current genotyping assays in distinguishing absent alleles from PCR failure.
  • To provide a versatile tool for marker-assisted selection and other genetic studies.

Main Methods:

  • Developed a multiplex Fluorescence-based Absent Allele-Specific Amplification (FAASA) method utilizing the PACE marker system.
  • Employed a primer mix including target-specific primer pairs (TSP) and core gene-specific primer pairs (CGSP).
  • Utilized 5' terminal tails on forward primers for compatibility with dye-labeled oligonucleotides in FRET cassettes.

Main Results:

  • FAASA successfully distinguishes between the absence of a target allele and a failed PCR reaction.
  • Lines amplifying only the core gene lack the target allele, while those amplifying both have the target allele.
  • The method provides a robust and affordable solution for high-throughput genotyping of presence/absence variation.

Conclusions:

  • FAASA offers a significant advancement for genotyping genes with presence/absence variation in crops.
  • FAASA markers are compatible with existing KASP and PACE workflows, facilitating integration into selection programs.
  • The method is adaptable for various genotyping applications, including GWAS, QTL, and bi-parental mapping studies across species.