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Determining 3′-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells
JoVE Journal
Genetics
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JoVE Journal Genetics
Determining 3′-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Determining 3′-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

DOI:
10.3791/58715-v

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13:07 min

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January 30, 2019

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, ,
1Department of Infectious Diseases, School of Immunology & Microbial Sciences,King’s College London

Chapters

  • 00:04Title
  • 00:32HIV-1 DNA Enrichment by Hybrid Capture
  • 03:54Adaptor Ligation
  • 06:03Adaptor Removal and Size Separation
  • 09:36DNA Extraction
  • 11:13Results: Characterization of Nascent Retroviral cDNAs within HIV-1 Infected Cells
  • 12:40Conclusion

Summary

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Here we present a deep sequencing approach that provides an unbiased determination of nascent 3'-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

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Keywords: HIV-1 Reverse TranscriptionNascent Single-stranded Viral DNA3′-terminiRetrovirologyCellular Restriction FactorsAntiretroviral DrugsHybrid CapturePCR WorkstationMagnetic Streptavidin BeadsBiotinylated OligonucleotidesDNA DenaturationDNA Hybridization

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