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Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase.

Jia Zhang1,2,3,4, Xiaolu Su1,2,3,4, Yefei Wang2,3,4,5

  • 1Single-Cell Center, CAS Key Laboratory of Biofuels, Shandong Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, China.

Frontiers in Bioengineering and Biotechnology
|July 17, 2023
PubMed
Summary

Improved Single-cell Genome Amplification (iSGA) enhances whole genome amplification for single-cell sequencing. This new method boosts efficiency and coverage, making large-scale single-cell sequencing more accessible and cost-effective.

Keywords:
GB1 fusion proteindisulfide bondphi29 DNA polymeraseprocess engineering high-efficiency phi29 DNA polymerasesingle-cell genome amplification

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Area of Science:

  • Genomics
  • Molecular Biology
  • Biotechnology

Background:

  • Single-cell genomic whole genome amplification (WGA) is essential for single-cell sequencing.
  • Current WGA methods suffer from low efficiency, incomplete, and uneven genome amplification, limiting throughput.
  • These limitations hinder the efficiency of single-cell sequencing workflows.

Purpose of the Study:

  • To introduce an improved single-cell genome amplification (iSGA) process.
  • To enhance the efficiency and coverage of whole genome amplification for single-cell sequencing.
  • To develop a cost-effective and robust method for large-scale single-cell sequencing.

Main Methods:

  • Engineering phi29 DNA polymerase through disulfide bond formation (F137C-A377C) to improve amplification ability.
  • Further protein and process engineering to develop a superior enzyme, HotJa Phi29 DNA Polymerase.
  • Application of iSGA to commercial probiotic samples to assess performance.

Main Results:

  • HotJa Phi29 DNA Polymerase achieved 99.75% genome coverage at 40°C.
  • iSGA demonstrated high single-cell genome amplification ability and 93.59% coverage in probiotic samples.
  • iSGA showed 2.03-fold higher efficiency and was 10.89-fold cheaper than commercial alternatives.

Conclusions:

  • iSGA offers a more efficient and robust solution for single-cell whole genome amplification.
  • The engineered HotJa Phi29 DNA Polymerase significantly improves amplification coverage and efficiency.
  • iSGA holds promise for broad applications in large-scale single-cell sequencing due to its cost-effectiveness and performance.