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Related Concept Videos

PCR01:32

PCR

Overview
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.
Real Time RT-PCR02:57

Real Time RT-PCR

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.
The real-time quantification of the number of amplified products is...

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Related Experiment Video

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Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp
10:44

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Multiplex, fluorescent single-strand conformation polymorphism using stepped polymerase chain reaction primers.

R Scholl1, A Walker, L W Ballard

  • 1Genomics Core Facility, University of Utah, Salt Lake City, UT 84132, USA.

Journal of Biomolecular Techniques : JBT
|June 6, 2009
PubMed
Summary

A novel high-throughput screening method was developed for detecting APC gene mutations. This approach utilizes stepped PCR primers and pooled analysis for efficient mutation detection.

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Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

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Area of Science:

  • Genetics and Molecular Biology
  • Biotechnology

Background:

  • Accurate detection of mutations in the Adenomatous Polyposis Coli (APC) gene is crucial for understanding associated diseases.
  • Existing methods for mutation screening can be time-consuming and may lack efficiency for large-scale analysis.

Purpose of the Study:

  • To develop and validate a high-throughput screening method for identifying a specific, well-characterized mutation within the APC gene locus.
  • To enhance the efficiency and capacity of mutation detection assays.

Main Methods:

  • Development of a method employing stepped PCR primers for targeted amplification.
  • Utilization of pooled product analysis to increase sample throughput.
  • Application of fluorescent single-strand conformation polymorphism (SSCP) for sensitive mutation detection.
  • Validation using a panel of known normal, heterozygous, and homozygous mutant samples.

Main Results:

  • Successful development of eight distinct stepped PCR products exhibiting consistent mobility shifts on the PE Biosystems 373A platform.
  • Demonstration that the method can accurately differentiate between normal and mutant APC alleles.
  • Establishment of a protocol allowing for the pooling of up to eight different samples for simultaneous analysis in a single lane.

Conclusions:

  • The developed method offers a robust and efficient approach for high-throughput screening of APC gene mutations.
  • This technique significantly enhances sample throughput by enabling pooled analysis, making it suitable for large-scale genetic screening.
  • The fluorescent SSCP-based strategy provides a sensitive and reliable means for detecting specific genetic alterations.