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Seamless 클로닝 기법을 이용한 ABO 엑손 2부터 엑손 7을 포함하는 증폭산물 클로닝

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

대한수혈학회지 2020년 31권 3호 p.239 ~ 246
이석기, 박건,
소속 상세정보
이석기 ( Lee Seog-Ki ) - Chosun University College of Medicine Department of Thoracic and Cardiovascular Surgery
박건 ( Park Geon ) - Chosun University College of Medicine Department of Laboratory Medicine

Abstract


Background: Sequencing and allele separation from exon 2 to exon 7 in the ABO gene 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.

키워드

ABO; Long PCR; Seamless cloning; Sequencing

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