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Related Concept Videos

Comparing Copy Number Variations and SNPs02:26

Comparing Copy Number Variations and SNPs

Sequencing of the human genome has opened up several best-kept secrets of the genome. Scientists have identified thousands of genome variations that exist within a population. These variations can be a single nucleotide or a larger chromosomal variation.
Copy number variations or CNVs are the structural variations that cover more than 1kb of DNA sequence. The single nucleotide polymorphism (SNP), on the other hand, is a single nucleotide change or a point mutation that is found in more than 1%...
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

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Detection of Copy Number Alterations Using Single Cell Sequencing
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Published on: February 17, 2017

Absolute SILAC-compatible expression strain allows Sumo-2 copy number determination in clinical samples.

Ivan Matic1, Ellis G Jaffray, Senga K Oxenham

  • 1Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, DD1 5EH, Scotland, United Kingdom.

Journal of Proteome Research
|August 12, 2011
PubMed
Summary

Researchers developed a cost-effective method for producing labeled protein standards using genetically modified bacteria. This advance simplifies absolute protein quantitation by mass spectrometry, making it more accessible for life science research.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Proteomics

Background:

  • Quantitative mass spectrometry is crucial in life sciences.
  • Production of labeled protein standards for absolute quantitation is costly and inefficient.
  • Existing methods face challenges with labeled amino acid conversion.

Purpose of the Study:

  • To develop an optimized, low-cost method for producing isotopically labeled protein standards.
  • To engineer a bacterial strain for efficient expression of fully labeled proteins.
  • To facilitate wider adoption of absolute quantitation in mass spectrometry.

Main Methods:

  • Genetically modified Escherichia coli BL21 (DE3) to create an arginine and lysine auxotroph.
  • Achieved high-level expression of fully labeled proteins without arginine-to-proline conversion.
  • Utilized fluorescence-based quantitation and LC-MS/MS analysis of cell lysates.

Main Results:

  • Successfully produced low-cost, fully labeled protein standards.
  • Demonstrated accurate determination of small ubiquitin-like modifier (SUMO-2) copy numbers in various human cells.
  • Streamlined the generation of labeled standards for absolute quantitation.

Conclusions:

  • The engineered bacterial strain significantly improves the production of labeled protein standards.
  • This method enhances the feasibility and accessibility of absolute protein quantitation by mass spectrometry.
  • The improved production system is expected to broaden the application of absolute quantitation techniques.