Cloning of ABO PCR Products Including Exon 2 to Exon 7 Using Seamless Cloning Technique

Abstract

Background: Sequencing and allele separation from exon 2 to exon 7 in the ABO gene can be required to deter-mine the genotype. This study used the seamless cloning technique to clone approximately 7.3 kb containing a portion of exon 2 to exon 7 of the ABO gene. Methods: The previously confirmed DNA sample of the ABO*B.01/O.01 genotype was used for seamless cloning using the Overlap Cloner DNA Cloning Kit (ElpisBio). Cloning was performed three times using the same cloning protocol. The plasmids obtained by cloning were also confirmed by sequencing. Results: Ninety-two colonies (average of 30 colonies per culture plate) were obtained. Of the fifty colonies, 48 colonies were properly amplified. Plasmid sequencing confirmed that 31 heterozygous alleles were well separated in accordance with predictions through the BGMUT database. Conclusion: The data suggest that the seamless cloning technique is a reliable technique that can be used to separate long-range alleles, such as the ABO gene. (Korean 2020;31:239-246)