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Related Concept Videos

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...
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.
PCR - Polymerase Chain Reaction01:32

PCR - Polymerase Chain Reaction

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RNA-seq03:21

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Southern Blot02:57

Southern Blot

Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
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PCR01:32

PCR

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Split Hybridization Probe Utilizing a DNA Fluorescent Light-up Aptamer as a Signal Reporter for Sequence-Specific Nucleic Acid Analysis
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Split Hybridization Probe Utilizing a DNA Fluorescent Light-up Aptamer as a Signal Reporter for Sequence-Specific Nucleic Acid Analysis

Published on: July 8, 2025

Ligation with nucleic acid sequence-based amplification.

Carmichael Ong1, Warren Tai, Aartik Sarma

  • 1Center for Biomedical Engineering, School of Engineering and Division of Biology and Medicine, Brown University, Providence, RI 02912, USA.

The Journal of Molecular Diagnostics : JMD
|March 28, 2012
PubMed
Summary
This summary is machine-generated.

This novel nucleic acid detection method uses a ligation step and isothermal amplification. The assay accurately identifies targets, even with single nucleotide changes, showing promise for diagnostic applications.

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Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA
11:58

Linear Amplification Mediated PCR – Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Published on: June 25, 2014

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Nucleic Acid Detection

Background:

  • Accurate detection of nucleic acid targets is crucial for diagnostics.
  • Existing methods may face challenges with sensitivity and specificity.
  • Isothermal amplification offers advantages for point-of-care applications.

Purpose of the Study:

  • To develop a novel method for nucleic acid detection.
  • To utilize a ligation step combined with isothermal, exponential amplification.
  • To assess the assay's specificity and sensitivity, including performance with single nucleotide polymorphism (SNP) targets.

Main Methods:

  • Design of an engineered single-stranded DNA (ssDNA) probe with two variable regions.
  • Ligation of the two-part probe by T4 DNA Ligase upon binding to the target sequence.
  • Isothermal, exponential amplification of the ligated product.
  • Optimization of reaction conditions, including KCl concentration and probe ratios.
  • Testing with synthetic targets and SNP variants.

Main Results:

  • The assay successfully detected the target nucleic acid, producing a specific 72-nt RNA product.
  • An extraneous 38-nt RNA product from linear amplification did not lead to false positives.
  • Optimal KCl concentration was determined to be 40 mmol/L.
  • Increasing the concentration of one probe fragment (P5) relative to another (P3) improved ligation and reduced the extraneous product.
  • The assay demonstrated high specificity, yielding a negative signal for a target with a single base change (SNP).
  • Using probe fragments with longer binding sites enhanced overall assay sensitivity.

Conclusions:

  • The developed ligation-mediated isothermal amplification assay is a novel and effective method for nucleic acid detection.
  • The assay exhibits high specificity and sensitivity, capable of distinguishing single nucleotide variations.
  • The method holds significant potential for various diagnostic applications.