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Related Concept Videos

The Replisome03:01

The Replisome

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DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with...
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Proofreading01:31

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Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
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During replication, the complementary strands in double-stranded DNA are synthesized at different rates. Replication first begins on the leading strand. Replication starts later, occurs more slowly, and proceeds discontinuously on the lagging strand.
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DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
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Homologous Recombination02:31

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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation
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Design Principles for Polymerase Strand Recycling Circuits.

Yueyi Li1,2, Arno E Gundlach3, Andrew D Ellington3,4

  • 1Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, USA.

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This study enhances cell-free biosensor technology using Polymerase Strand Recycling (PSR) circuits for improved microRNA detection. Engineered PSR systems offer greater speed, sensitivity, and specificity in diagnostic applications.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Synthetic Biology

Background:

  • Cell-free biosensing systems offer programmable diagnostic capabilities.
  • Molecular circuits enhance biosensor performance through computation and signal amplification.
  • Polymerase Strand Recycling (PSR) is a previously developed circuit for amplifying cell-free molecular systems.

Purpose of the Study:

  • To generalize the Polymerase Strand Recycling (PSR) platform for detecting diverse microRNA inputs.
  • To enhance PSR circuit function through engineering of T7 RNA polymerase (RNAP).
  • To provide troubleshooting strategies for optimizing PSR circuit performance.

Main Methods:

  • Development and engineering of PSR circuit design principles.
  • Utilizing T7 RNA polymerase (RNAP) for off-target transcription and nucleic acid input recycling.
  • Testing PSR circuits with a diverse set of model microRNA targets.
  • Engineering T7 RNAP to improve PSR circuit efficiency.

Main Results:

  • Demonstrated successful generalization of PSR circuits for detecting various microRNA targets.
  • Showcased enhanced PSR circuit function through T7 RNAP engineering.
  • Identified key strategies for optimizing PSR circuit performance and troubleshooting issues.

Conclusions:

  • The generalized PSR platform significantly expands the utility of cell-free biosensors for microRNA detection.
  • Engineering T7 RNAP offers a viable approach to boost PSR circuit efficiency.
  • Optimized PSR circuits represent a promising advancement in sensitive and specific molecular diagnostics.