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Related Concept Videos

Next-generation Sequencing03:00

Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.
Sanger Sequencing01:57

Sanger Sequencing

DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...

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Related Experiment Video

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Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria
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Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Published on: November 10, 2015

Nucleotide extension genotyping by high-resolution melting.

Michael Liew1, Carl Wittwer, Karl V Voelkerding

  • 1Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108-1221, USA. liewm@aruplab.com

The Journal of Molecular Diagnostics : JMD
|September 18, 2010
PubMed
Summary
This summary is machine-generated.

A novel single nucleotide extension assay coupled with high-resolution melting effectively genotypes challenging single nucleotide polymorphisms. This method offers a reliable alternative for genetic variation analysis, demonstrating high concordance across multiple genes.

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Area of Science:

  • Genetics
  • Molecular Biology
  • Biotechnology

Background:

  • Small amplicon melting has limitations in genotyping specific symmetric DNA variations.
  • A need exists for methods that can accurately genotype these challenging variations without complex sample manipulation.

Purpose of the Study:

  • To develop and validate a high-resolution melting single nucleotide extension assay for genotyping single nucleotide polymorphisms (SNPs).
  • To assess the performance of this assay on various genes, including RET, HFE, F2, F5, and MTHFR.

Main Methods:

  • Utilized a LightScanner 32 (LS32) instrument for real-time PCR and sequential high-resolution melting of 32 samples.
  • Employed asymmetric PCR with Platinum Taq and LC Green Plus, followed by post-PCR addition of dideoxynucleotides and extension oligonucleotides.
  • Analyzed DNA melting profiles to identify SNPs based on melting temperature transitions.

Main Results:

  • Successfully genotyped SNPs in RET, HFE, F2, F5, and MTHFR genes.
  • Achieved 100% concordance with previous genotyping for HFE, MTHFR, and F2.
  • Demonstrated 90% concordance for F5, with discordances resolved by assay redesign.

Conclusions:

  • The high-resolution melting single nucleotide extension assay is effective for differentiating SNPs.
  • Careful optimization is crucial for successful implementation and accurate genotyping.
  • This method provides a valuable tool for genetic variation analysis, particularly for challenging SNPs.