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Related Concept Videos

The DNA Replication Fork01:02

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An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.   Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication...
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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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DNA unwinding helicase enzymes are a type of motor protein. Motor proteins can translocate along filaments or polymers using energy generated from ATP hydrolysis. Helicases are involved in all the important cellular processes where DNA unwinding is required, such as DNA replication, repair, recombination, and transcription. They are present in all living organisms, but vary in their structure, function, and mechanism of action. For example, in prokaryotes, DnaB helicase binds and translocates...
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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.
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Related Experiment Video

Updated: Feb 27, 2026

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method
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Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

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Sequence motifs capable of forming DNA stem-loop structures act as a replication diode.

Andrey Shirak1, Uri Seroussi2, Elisha Gootwine1

  • 1Institute of Animal Science Agricultural Research Organization (ARO) Rishon Le Tsiyon Israel.

FEBS Open Bio
|July 7, 2017
PubMed
Summary

Sequencing DNA in different directions can yield different results due to a "replication diode" effect caused by DNA secondary structures. This bias can be minimized by lowering DNA and salt concentrations during sequencing for accurate copy number proportion estimation.

Keywords:
DNA loop structureDNA stabilityDNA thermodynamicsmicroRNAorientation‐dependent block

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Area of Science:

  • Molecular Biology
  • Genetics
  • Bioinformatics

Background:

  • Estimating copy number proportions (CNPs) relies on peak-height ratios in sequencing chromatograms.
  • An unexplained orientation bias in sequencing results has been observed.

Purpose of the Study:

  • Investigate the cause of orientation bias in sequencing DNA.
  • Identify DNA secondary structures influencing peak-height ratios.
  • Propose methods to minimize sequencing bias for accurate CNP estimation.

Main Methods:

  • Comparative analysis of Sanger and DNA-seq for specific SNPs.
  • High-resolution melt and MFOLD analyses to study DNA secondary structures.
  • Oligonucleotide and amplicon-based experiments to test hypotheses.
  • Bidirectional sequencing under varying ionic strengths.

Main Results:

  • A stem-loop structure with G-T pairing was identified as the cause of orientation bias.
  • This 'replication diode' effect hinders DNA polymerase on one strand.
  • Lowering DNA and salt concentration reduced the replication diode effect.
  • Genomic palindromes can also induce this effect.

Conclusions:

  • DNA secondary structures, specifically stem-loops, create an orientation bias in sequencing.
  • The 'replication diode' effect impacts accurate CNP estimation.
  • Optimizing ionic strength and employing bidirectional sequencing are crucial for reliable results.