2.2. DNA Constructs and Site-Directed Mutagenesis

To obtain DNA PCR calibrators, we used the pBH10 plasmid (obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: Human Immunodeficiency Virus 1 (HIV-1) BH10 Non-Infectious Molecular Clone (pBH10), ARP-90, contributed by Dr. Beatrice Hahn and Dr. George M. Shaw), which was diluted to the required concentrations using a series of 10-fold dilutions.

To obtain a control DNA construct, a target fragment of the HIV genome region, including the HIV integrase gene 412 bp long, was generated via PCR using specific primers: Int_for4 CCCTACAATCCCCAAAGTCARGGAGT and Int_rev5 CATCACCTGCCATCTGTTTTTCCATARTC and plasmid pBH10 as a DNA template.

The resulting amplicon was ligated into the pGEM vector (Promega, Madison, WI, USA). The ligase mixture was introduced through transformation into E. coli cells, strain DH10b. Screening of the obtained clones was carried out using the PCR method. The structure of the target fragment was confirmed using Sanger sequencing.

To obtain DNA constructs with mutations in the binding sites of the primers and probe, the control DNA construct was used, into which mutations were introduced using the QuikChange II Site-Directed Mutagenesis kit (Agilent, Santa Clara, CA, USA) in accordance with the manufacturer’s recommendations. The resulting DNA constructs were verified via Sanger sequencing.