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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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Resolving Breakpoints of Chromosomal Rearrangements at the Nucleotide Level Using Sanger Sequencing.

Katarena Nalbandian1,2,3, Raul E Piña-Aguilar2,4,3, Cynthia C Morton2,4,5,6,7

  • 1Massachusetts College of Pharmacy and Health Sciences University, Boston, Massachusetts.

Current Protocols in Human Genetics
|December 28, 2020
PubMed
Summary
This summary is machine-generated.

This study introduces a method to precisely pinpoint chromosomal breakpoints using Sanger sequencing after genome sequencing. This technique achieves nucleotide resolution, improving the interpretation of gene disruptions caused by structural variants.

Keywords:
Sanger sequencingchromosomal rearrangementscytogeneticsinversionstranslocations

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Area of Science:

  • Genetics
  • Genomics
  • Molecular Biology

Background:

  • Cytogenetic tools increasingly rely on genome sequencing for detecting chromosomal abnormalities.
  • Current sequence-based methods for structural variants lack nucleotide resolution for breakpoint interpretation.
  • Accurate breakpoint characterization is crucial for understanding gene disruption in chromosomal abnormalities.

Purpose of the Study:

  • To present a protocol for characterizing structural variants at nucleotide resolution.
  • To enable precise localization of chromosomal breakpoints for improved diagnostic interpretation.
  • To integrate Sanger sequencing with long-molecule genome sequencing methods.

Main Methods:

  • Primer design for junction amplification at translocations and inversions.
  • Amplification of derivative chromosomes using long-range or hot-start polymerase.
  • DNA preparation for Sanger sequencing.
  • Interpretation and reporting of breakpoints using Sanger sequencing data.

Main Results:

  • The protocol allows for the characterization of structural variants at nucleotide resolution.
  • Enables precise identification of breakpoints in chromosomal abnormalities.
  • Provides a method to overcome limitations of existing sequence-based techniques.

Conclusions:

  • This protocol offers a reliable method for nucleotide-level breakpoint characterization.
  • Enhances the diagnostic utility of genome sequencing for chromosomal abnormalities.
  • Facilitates accurate interpretation of gene disruption resulting from structural variants.