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Related Concept Videos

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...
DNA Isolation01:24

DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
PCR01:32

PCR

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DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation
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Padlock probe-mediated qRT-PCR for DNA computing answer determination.

Fusheng Xiong1, Wayne D Frasch

  • 1Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, P.O. Box 874501, Tempe, AZ 85287-4501, USA.

Natural Computing
|June 22, 2011
PubMed
Summary
This summary is machine-generated.

Padlock probe-quantitative real-time PCR (PLP-qRT-PCR) accurately quantifies short DNA sequences for DNA computing. This method enables sensitive, simultaneous detection of multiple DNA targets, advancing biotechnology applications.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Computational Biology

Background:

  • Quantitative DNA sequence detection is crucial for applications like DNA computing.
  • Existing methods may lack sensitivity or multiplexing capabilities for short DNA sequences.

Purpose of the Study:

  • To adapt padlock probe-mediated quantitative real-time PCR (PLP-qRT-PCR) for quantifying short DNA sequences.
  • To assess the efficiency and sensitivity of PLP-qRT-PCR for DNA computing applications.
  • To enable simultaneous quantification of multiple DNA targets.

Main Methods:

  • Designed linear padlock probes (PLPs) with specific 10-mer detection arms and universal PCR primers.
  • Utilized enzymatic ligation for target-dependent PLP circularization.
  • Employed quantitative real-time PCR (qRT-PCR) to amplify circularized PLPs, removing non-circularized templates.

Main Results:

  • Achieved high amplification efficiency (98.7%) for PLPs.
  • Demonstrated a linear detection range from 0.2 pg to 20 ng of target DNA.
  • Showed no significant difference in C(t) values between multiplex and singleplex assays for three targets.

Conclusions:

  • PLP-qRT-PCR offers a highly sensitive and efficient method for simultaneous quantification of multiple short nucleic acid sequences.
  • The protocol is suitable for DNA computing applications, such as solving the traveling salesman problem.
  • This technique has broad potential applications across various fields in biotechnology.