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Related Concept Videos

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...

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A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools
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A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

Published on: August 21, 2019

A simple and efficient method for isolating polymorphic microsatellites from cDNA.

Gen Hua Yue1, Ze Yuan Zhu, Chun Ming Wang

  • 1Molecular Population Genetics Group, Temasek Life Sciences Lab, 1 Research Link, National University of Singapore, 117604 Singapore. genhua@tll.org.sg

BMC Genomics
|March 27, 2009
PubMed
Summary
This summary is machine-generated.

We developed a simple method to isolate microsatellites from fish cDNA, significantly increasing efficiency. This technique aids in genetic mapping and understanding fish genome evolution.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Fish Biology

Background:

  • Microsatellites in cDNA are valuable molecular markers for gene transcription, linkage mapping, comparative mapping, and genome evolution studies.
  • Existing methods for microsatellite detection in fish cDNA are limited by the scarcity or absence of expressed sequence tags (ESTs).
  • Fishes constitute a significant portion of vertebrate biodiversity, highlighting the need for efficient genetic tools.

Purpose of the Study:

  • To develop a simple and efficient method for isolating microsatellites from fish cDNA.
  • To enhance the efficiency of microsatellite discovery in fish species with limited EST data.

Main Methods:

  • cDNA normalization using duplex-specific nuclease (DSN) treatment.
  • Enrichment of microsatellites using biotinylated oligonucleotides and magnetic separation.
  • Directional cloning of enriched cDNA into a vector for microsatellite isolation.

Main Results:

  • The developed method significantly increased microsatellite isolation efficiency by over 30 times compared to direct sequencing.
  • Out of 384 sequenced clones, 139 contained microsatellites, with 91 unique sequences.
  • Forty-one polymorphic microsatellites were characterized, showing an average of 4.85 alleles and 0.56 expected heterozygosity, with Mendelian inheritance.

Conclusions:

  • cDNA normalization substantially boosts the efficiency of microsatellite enrichment.
  • The described method is applicable to a wide range of fish species for microsatellite isolation.
  • Isolated microsatellites are valuable for fish linkage and comparative mapping, and for studying genome evolution.