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Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization
Published on: April 14, 2016
Quencher extension for single nucleotide polymorphism quantification in bacterial typing and microbial community
Knut Rudi1, Monika Zimonja, Beate Skånseng
1Matforsk AS, Norwegian Food Research Institute, Oslo, Norway.
Methods in Molecular Biology (Clifton, N.J.)
|September 8, 2006
Summary
This study introduces quencher extension, a new real-time method for quantifying single nucleotide polymorphisms (SNPs). This technique measures fluorescence quenching to determine SNP allele amounts in microbial samples.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Quantifying single nucleotide polymorphisms (SNPs) is crucial for genetic analysis.
- Existing methods may require multiple steps or open-tube reactions.
- There is a need for efficient and closed-tube SNP quantification techniques.
Purpose of the Study:
- To develop and present a novel single-step, closed-tube real-time method for SNP quantification.
- To demonstrate the utility of the quencher extension method for microbial analyses.
Main Methods:
- The quencher extension method utilizes a probe with a 5'-reporter and a quencher.
- Single-base extension with a dideoxy nucleotide occurs if the target SNP allele is present.
- Fluorescence quenching of the reporter is measured in real-time during thermocycling.
Main Results:
- The method allows for the quantification of specific SNP alleles based on nucleotide incorporation rates.
- Relative amounts of SNP alleles can be determined accurately.
- The protocol was successfully applied to SNP quantification in Listeria monocytogenes.
Conclusions:
- Quencher extension is an effective real-time method for quantifying SNPs in a single, closed-tube reaction.
- This technique is suitable for microbial community analyses and pathogen detection.
