In the first infected group of mice, after a month, the mice were killed and the CD4+ T cells were purified from the spleen to be sequenced. They extracted the DNA using the MasterPureTM DNA purification kit and then subjected the CCR5 ZFN binding site to PCR amplification for 25 cycles. After PCR amplification and gel purification the sequence was subject to a modified Surveyor nuclease assay to determine CCR5 disruption frequency. Surveyor nuclease assays are used to detect single base mismatches or small insertions or deletions. It uses a mismatch-specific endonuclease that recognizes all base substitutions and insertions or deletions. When it finds one of the three, it cleaves the 3’ side of the mismatched side in both of the DNA strands. This process consists of five steps, DNA extraction, PCR, formation of hybrid DNA duplex, digestion,
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This was conducted on the second group of infected mice. Blood samples from the mice were taken each day. The blood was analyzed with another Surveyor assay to measure CCR5 gene disruption and then western blotting to compare HIV-1 viral RNA loads. A positive result that supports the hypothesis of the CCR5 ZFN-disruption would be bands on the Surveyor nuclease assay that travel further down the gel. This indicates that the mismatch was present and the nuclease correctly excised it from the DNA, resulting in a new band. This excision would mimic the naturally occurring mutation and provide an immunity to HIV-1. A negative result that refutes the CCR5 ZFN-disruption would be a Surveyor assay result containing one single band, which would indicate that no mismatch was present, and that the HIV-1 virus was not inhibited by the ZFN. The results found from this experiment were displayed in Figure 4. of the paper and are shown below in Figure 1. (Perez et
This was conducted on the second group of infected mice. Blood samples from the mice were taken each day. The blood was analyzed with another Surveyor assay to measure CCR5 gene disruption and then western blotting to compare HIV-1 viral RNA loads. A positive result that supports the hypothesis of the CCR5 ZFN-disruption would be bands on the Surveyor nuclease assay that travel further down the gel. This indicates that the mismatch was present and the nuclease correctly excised it from the DNA, resulting in a new band. This excision would mimic the naturally occurring mutation and provide an immunity to HIV-1. A negative result that refutes the CCR5 ZFN-disruption would be a Surveyor assay result containing one single band, which would indicate that no mismatch was present, and that the HIV-1 virus was not inhibited by the ZFN. The results found from this experiment were displayed in Figure 4. of the paper and are shown below in Figure 1. (Perez et