HIV-1 DNA的量化

Ferdinand Roesch; Olivier Schwartz

doi:10.21769/BioProtoc.417

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Peer-reviewed

Quantification of HIV-1 DNA

HIV-1 DNA的量化

FR Ferdinand Roesch

OS Olivier Schwartz email

发布: 2013年04月05日第3卷第7期 DOI: 10.21769/BioProtoc.417 浏览次数: 12053

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Jul 2012

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Abstract

The reverse transcription (RT) reaction is a critical step in HIV-1 life cycle. It is very strongly regulated and the target of several restriction factors (TRIM5α, APOBECs, SAMHD1, etc.). The progress of reverse transcription can be followed by measuring viral DNA by quantitative PCR (qPCR). This method is sensitive enough to allow detection of low amounts of HIV-1 DNA in infected cells and discriminate between several types of reverse transcription intermediates (so called 《early》 and 《late》 RT products, 2 Long Terminal Repeat (LTR) circles, integrated DNA).

Materials and Reagents

Cells of interest, infected with HIV-1
Proteinase K (20 mg/ml) (> 30 U/mg) (Eurobio, catalog number: GEXPRK01-B5 )
AL buffer (QIAEN, catalog number: 19075 )
Phenol-chloroform – isoamyl alcohol (Sigma-Aldrich, catalog number: 77617-100 ml )
Chloroform (Sigma-Aldrich)
Pure water (Life Technologies, Gibco^®)
Absolute Ethanol (Sigma-Aldrich)
3 M sodium acetate (pH 5.0)
DpnI Fast Digest restriction enzyme and reaction buffer (Fermentas, catalog number: FD1704 )
Platinium SYBR Green qPCR Supermix UDG (Life Technologies, Invitrogen^TM, catalog number: 11733-046 )
qPCR primers
Standard plasmids for HIV-1 (pNL4-3) and GAPDH (pGAPDH)

Equipment

Centrifuge
DNA-free hood
Speed-vac
Heat-seal machine
qPCR machine
96-well qPCR plates (Eppendorf)
Plastic films for qPCR plates (Eppendorf)
Filter tips

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Roesch, F. and Schwartz, O. (2013). Quantification of HIV-1 DNA. Bio-protocol 3(7): e417. DOI: 10.21769/BioProtoc.417.
Roesch, F., Meziane, O., Kula, A., Nisole, S., Porrot, F., Anderson, I., Mammano, F., Fassati, A., Marcello, A., Benkirane, M. and Schwartz, O. (2012). Hyperthermia stimulates HIV-1 replication. PLoS Pathog 8(7): e1002792.