Genomic DNA from whole blood was isolated using a genomic DNA isolation kit according to the manufacturer’s instructions (MACHEREY-NAGEL, Germany). Genomic DNA was diluted, and a polymerase chain reaction (PCR) was performed. The thermal conditions for PCR were programmed. The amplified PCR products were resolved on 2% agarose gel electrophoresis; the gel was excised, and its size was minimized. It was, then, extracted using QIAquick Gel Extraction Kit. The amplified PCR products were eluted in TE buffer and sequenced by the Sanger sequencing method (Applied Biosystems). After DNA sequencing, we analyzed the electropherograms of the two SNPs located within exon 4 of the ApoE gene, rs429358, and rs7412, and categorized the alleles for ε2/ε2, ε2/ε3, ε2/ε4, ε3/ε3, ε3/ε4, and ε4/ε4, respectively. Participants were categorized based on the number of ε4 alleles (0, 1, or 2) into three groups: No ε4 (ε2/ε2, ε3/ε3, and ε2/ε3), heterozygous ε4 (ε2/ε4 and ε3/ε4), and homozygous ε4 (ε4/ε4).
Sanger sequencing method
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Sanger sequencing method is a technique used for determining the nucleotide sequence of DNA molecules. It relies on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. This process generates a set of DNA fragments of varying lengths that can be separated by gel electrophoresis and analyzed to determine the original DNA sequence.
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Lab products found in correlation
2 protocols using sanger sequencing method
1
Genotyping ApoE Polymorphisms
2
Molecular Identification of Piroplasma spp.
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A group of purified DNA samples that tested positive for Piroplasma spp. by cPCR and negative by mPCR was further screened by nPCR using primers targeting the hypervariable (V4) region of the 18S rRNA gene. The primer sequences were used for external PCR reaction [RLB-F2 and RLB-R2] and nPCR [RLB-FINT and RLB-R2]. The PCR external and internal reaction conditions were set up according to Liu et al. (24 (link)). The PCR products obtained from representative positive samples by nPCR were purified using ExoSAP-IT reagent (Applied Biosystems, Lithuania, North-eastern Europe) and then sequenced (Sanger sequencing method, Eurofins Genomics, SimpleSeq service, Louisville, KY, United States).
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