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The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...
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Related Experiment Video

Updated: Feb 13, 2026

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
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Double-Stranded RNA-Enriched Preparations to Identify Viroids by Next-Generation Sequencing.

Beatriz Navarro1, Francesco Di Serio2

  • 1Istituto per la Protezione Sostenibile delle Piante, Consiglio Nazionale delle Ricerche, Bari, Italy.

Methods in Molecular Biology (Clifton, N.J.)
|March 2, 2018
PubMed
Summary

This study presents a new protocol for detecting plant viroids using next-generation sequencing (NGS). The method efficiently identifies viroid infections in citrus plants by analyzing double-stranded RNA from bark samples.

Keywords:
CitrusNGSViroidsdsRNA

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Area of Science:

  • Plant Pathology
  • Molecular Biology
  • Bioinformatics

Background:

  • Viruses and viroids pose significant threats to plant health, impacting agriculture and ecosystems.
  • Accurate detection methods are crucial for managing plant diseases and preventing spread.
  • Existing methods for viroid detection can be limited in scope or efficiency.

Purpose of the Study:

  • To develop and describe an efficient protocol for nucleic acid extraction and enrichment from citrus bark.
  • To enable the identification of viroids infecting citrus plants using next-generation sequencing (NGS) and bioinformatics.
  • To provide a reliable method for diagnosing viroid diseases in woody plants.

Main Methods:

  • Nucleic acid extraction from citrus bark tissue.
  • Enrichment of double-stranded RNA (dsRNA) from extracted nucleic acids.
  • Construction of complementary DNA (cDNA) libraries.
  • Pair-end sequencing of cDNA libraries.
  • Bioinformatics analysis for viroid identification.

Main Results:

  • The protocol successfully extracts and enriches dsRNA from citrus bark.
  • Generated cDNA libraries are suitable for high-throughput sequencing.
  • Bioinformatics analysis effectively identifies known and potentially novel viroids in the samples.
  • The method demonstrates high efficiency in detecting viroid infections.

Conclusions:

  • This protocol offers an efficient and reliable approach for viroid detection in citrus and potentially other woody plants.
  • The integration of NGS and bioinformatics provides a powerful tool for plant viroid diagnostics.
  • This method can aid in disease management and epidemiological studies of plant viroids.