Parasite DNA was analysed on day 0 for the presence of mutations in the Pfk13 propeller domain, which is associated with artemisinin resistance. The propeller domain was amplified in a nested-PCR assay, amplicons were sequenced according to Sanger’s method (Macrogen, Republic of Korea), and DNA sequences were analysed to identify specific single nucleotide polymorphisms (SNPs) related to artemisinin resistance [9 (link)]. The amino acid sequences were compared with the 3D7 wild-type amino acid sequences PF3D7_1343700. The presence of SNPs was confirmed by reading both the forward and the reverse strands. Parasites with mixed alleles were considered mutants. The number of copies of Pfpm2 genes was assessed by reverse transcriptase-PCR (sybr green dye). The full method has been described by Witkowski et al. [10 (link)].
Sanger s method
Manufactured by Macrogen
Sanger's method is a DNA sequencing technique used to determine the order of nucleotides in a DNA molecule. It is a reliable and commonly used method for DNA sequencing. The core function of Sanger's method is to generate a set of DNA fragments with different lengths, each terminating with a specific nucleotide, which can then be separated and analyzed to determine the DNA sequence.
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Lab products found in correlation
2 protocols using sanger s method
1
Genetic Analysis of Artemisinin Resistance
2
Cloning and Sequencing of Amplicons
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For each isolate, amplicons scattered in the 450–700-bp region were gel-purified, ligated into the pGEM-T Easy Cloning Vector Kit (Promega, Madison, WI, USA), and transformed into Escherichia coli DH5α competent cells according to the manufacturer’s instructions. Twenty to forty colonies per each cloned amplicon were selected and sequenced using Sanger’s method (Macrogen Inc., Daejeon, Korea). The obtained sequences were aligned with global sequences from GenBank and analyzed using CLC Main Workbench 6.
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