The video demonstrates the procedure for a growth competition assay to measure the relative replication fitness of two viral strains by comparing their replication in human immune cells.
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Method Article
An Assay for Measuring the Relative Replication Fitness of Viral Strains
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July 31st, 2026
In This Article
Abstract
Protocol
1. Construction of Chimeric HIV-1 NL4-3 Molecular Clones
- Amplify Insert DNA Fragment
- Design chimeric primers. The 5’ halves of both forward and reverse primers contain an HIV-1 vector sequence, at which the fragment will be inserted. The 3’ half of the primers must contain the end of the insert sequence (Figure 1). Make sure that the chimeric primer sequence retains the original open reading frames.
- Use primers at least 20 bases in length, with a melting temperature greater than or equal to 60 °C, ~50% GC content, and a low tendency to form primer dimers, heterodimers, and/or hairpin structures. ....
- Design chimeric primers. The 5’ halves of both forward and reverse primers contain an HIV-1 vector sequence, at which the fragment will be inserted. The 3’ half of the primers must contain the end of the insert sequence (Figure 1). Make sure that the chimeric primer sequence retains the original open reading frames.
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Results
Figure 1: Construction of HIV-1 NL4-3 COTB Gag-p24 recombinant molecular clones using overlap extension PCR. (A) Design the chimeric primers. The 5’ halves of the primers contain the NL4-3 vector sequence and the 3&#x.......
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Construction of recombinant clones | |||
| Chimeric primer/Mutagenic/Sequencing primers | IDT | Custom DNA oligos | |
| pNL4-3VifA and pNL4-3VifB plasmid | |||
| High-Fidelity DNA polymerase | Thermo Scientific | F-549S | |
| High-fidelity buffer | Thermo Scientific | F-549S | |
| Deoxynucleotide triphosphate (dNTP) | Bioline | Bio-39026 | |
| Thermal cycler | Life Technologies | Applied Biosystems® GeneAmp® PCR System 9700 | |
| DNA loading dye | Thermo Scientific | R0631 | |
| 1 kb Plus DNA ladder | Invitrogen | 10787-018 | |
| QIAquick PCR Purification Kit | Qiagen | 28104 | |
| QIAquick gel extraction kit | Qiagen | 28704 | |
| DpnI enzyme | New England Biolabs | R0176S | |
| TOP10 chemically competent Escherichia coli | Invitrogen | C4040-10 | |
| Luria Broth base powder | Invitrogen | 12795-084 | |
| Carbenicillin | Research Products International | C46000-25.0 | |
| QIAprep Spin Miniprep kit | Qiagen | 27104 | |
| EndoFree Plasmid Maxi Kit | Qiagen | 12362 | |
| Transfection | |||
| X-tremeGENE 9 DNA transfection reagent | Roche | 6365787001 | |
| HEK 293T-17 | ATCC | CRL-11268 | http://www.atcc.org/ |
| 0.22 μm filter top tube | VWR International | 89220-716 | |
| Cell culture | |||
| Dulbecco's Modified Eagle Medium (DMEM) | Life Technologies | 10566016 | |
| Iscove′s Modified Dulbecco′s Medium (IMDM) | Life Technologies | 31980-030 | |
| RPMI-1640 media | Life Technologies | 61870-036 | |
| Phytohemagglutinin (PHA) | Thermo Scientific | R30852801 | |
| Human Interleukin-2 | Roche | 11147528001 | |
| Fetal bovine serum | JR Scientific | 43640 | |
| Penicillin and Streptomycin | Corning Cellgro | 30-001-CI | |
| 6 well plate | VWR Scientific | 73520-906 | |
| 48 well plate | VWR Scientific | 62407-338 | |
| 96 well flat-bottomed plate | ISC Bioexpress | T-3015-4 | |
| 96 well round-bottomed plate | BD Falcon | 353077 | |
| 1.5 ml Microcentrifuge Tube | Mt. Baker Bio | MBD-1500 | |
| 1.5 ml tubes w/ O-ring | VWR Scientific | 89004-290 | |
| 50 ml conical tube | ISC Bioexpress | C-3317-6 |
