Korean J Blood Transfus.  2020 Dec;31(3):239-246. 10.17945/kjbt.2020.31.3.239.

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

• Lee SK
• Park G

• KMID: 2509833
• DOI: http://doi.org/10.17945/kjbt.2020.31.3.239

Abstract

Background
Sequencing and allele separation from exon 2 to exon 7 in the ABOgene can be required to determine 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.

Keyword

ABO; Long PCR; Seamless cloning; Sequencing