Viral RNA was extracted from plasma samples obtained from MSM subjects with the QIAamp Viral RNA Mini-kit (Qiagen, UK). The Gag gene was amplified using the SuperScript Polymerase One-Step RT-PCR System (Takara, Dalian, China), followed by a second round of PCR with Gag-specific primers [36 (link)]. PCR products were gel purified with the QIAquick gel extraction kit (Qiagen, UK) and cloned with a TOPO TA cloning kit (Invitrogen, USA). The cloning PCR fragments were then sequenced with Sanger sequencing method using ABI 3730 DNA analyzer by Huada Genomics Company (China). A sequencer program was used to assemble and edit individual sequence fragments.
Superscript polymerase one step rt pcr system
Manufactured by Takara Bio
Sourced in China
The SuperScript Polymerase One-Step RT-PCR System is a laboratory equipment product that enables the reverse transcription and amplification of RNA in a single step. It utilizes a specialized reverse transcriptase enzyme and a high-fidelity DNA polymerase to facilitate the process.
Automatically generated - may contain errors
3 protocols using superscript polymerase one step rt pcr system
1
Viral RNA Extraction and Gag Gene Amplification
2
Amplification and Sequencing of HIV-1 nef Gene
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The entire nef gene was amplified using the SuperScript Polymerase One-Step RT-PCR System (Takara, Dalian, China) followed by nested PCR. The outer primers were O1 (5′-GTGCCTCTTCAGCTACCACCG-3′, 8513–8533 of HXB2) and O2 (5′-AGCATCTGAGGGTTAGCCACT-3′, 9488–9508 of HXB2). The first round of PCR was performed with the following parameters: 56 °C for 30 min; 94 °C for 5 min; followed by 3 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 2 min and 32 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min; and a final extension step at 72 °C for 10 min. The inner primers were I1 (5′-TGGACAGATAGGGTTATAGAA-3′, 8697–8717 of HXB2) and I2 (5′-CACCTCCCCTGGAAAGTCCCC-3′, 9448–9468 of HXB2). The conditions for the second round of PCR were as follows: 94 °C for 5 min; 3 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 2 min and 32 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min; and a final extension step at 72 °C for 10 min. The PCR products were purified using the QIAquick Gel Extraction Kit (Qiagen) and cloned using a TOPO TA cloning kit (Invitrogen, USA). The fragments were sequenced by Huada Genomics Company (Beijing, China). The Sequencher program (version 4.9) was used to assemble and edit the sequence fragments.
3
Viral RNA Extraction and Amplification for HIV-1 Analysis
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Viral RNA was extracted from plasma using the QIAamp Viral RNA Mini-kit (Qiagen, UK) according to the manufacturer's instructions. The entire gag gene was amplified with the SuperScript Polymerase One-Step RT-PCR System (Takara, Dalian, China). The first round of PCR with outer primers 172A (5′-ATCTCTAGCAGTGGCGCCCGAACAG-3′ 628–648 nt of HIV-1 HXB2) and Gag-6 (5′-TAATGCTTTTATTTTYTCTTCTGTCAATGGC-3′ 2651–2621 nt of HIV-1 HXB2) was performed with the following cycling parameters: 56°C for 30 min; 94°C for 5 min; followed by 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 2.5 min; and a final extension step at 72°C for 10 min. The second round of PCR with inner primers Gag-763 (5′-TGACTAGCGGAGGCTAGAAGG-3′ 763–783 nt of HIV-1 HXB2) and Gag-5 (5′-TTCCYCCTATCATTTTTGGTTTCC-3′ 2377–2400 nt of HIV-1 HXB2) was performed with the following cycling parameters: 94°C for 5 min; followed by 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 2.5 min; and a final extension step at 72°C for 10 min. The PCR products were confirmed through 1.0% agarose gel electrophoresis. PCR products were purified using the QIAquick gel extraction kit (Qiagen) and cloned using a TOPO TA cloning kit (Invitrogen, USA). The fragments were sequenced by Huada Genomics Company (China). Individual sequence fragments were assembled and edited using the Sequencher program (version 4.9).
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