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Evaluating novel effective primers to amplify heterozygous alleles of second, third and fourth exons of HLA-A; -B; -C; -DRB1 and -DQB1 loci using sequencing-based typing

Authors:

PCD Perera,

University of Colombo, LK
About PCD
Department of Chemistry, Faculty of Science
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BDN Upamali,

University of Colombo, LK
About BDN
Department of Chemistry, Faculty of Science
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YINS Gunawardene,

University of Kelaniya, LK
About YINS
Molecular Medicine Unit, Faculty of Medicine
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RS Dassanayake

University of Colombo, LK
About RS
Department of Chemistry, Faculty of Science
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Abstract

Human leukocyte antigen (HLA) typing is one of the most crucial steps that determines the success of an organ transplant. However, HLA typing is a challenging task due to the diversity of HLA alleles, which is caused by high polymorphism of the region and high number of guanine and cytosine bases that limits the degree of amplification. Low resolution serology typing currently employed in Sri Lanka may fail to identify subtle differences in certain alleles, which may affect the long-term survival of the organ recipient. Therefore, a low cost, high-resolution DNA-based typing method for the HLA loci of Sri Lankans was developed based on polymerase chain reaction (PCR) amplification followed by Sanger sequencing, which is considered to be the gold standard for HLA typing. With minimised PCR bias and equal chances of amplifying all the alleles curated so far, a novel set of primers were designed to amplify the second and third exons of alleles in group specific PCR. To increase the resolution of alleles further, the fourth exon was also amplified using novel primers designed in this study and primers reported in the literature. Touchdown PCR and hot-start PCR were used to optimise PCR conditions so that non-specific amplifications are minimal. SBTengine® (version 3.12.0.2724) software was used in assigning the sequence chromatogram to the allele sequence. Seventeen new primers were designed in this study to ensure the amplification and identification of both alleles in heterozygous individuals that were previously unable to be identified using primers reported in the literature.
How to Cite: Perera, P., Upamali, B., Gunawardene, Y. and Dassanayake, R., 2019. Evaluating novel effective primers to amplify heterozygous alleles of second, third and fourth exons of HLA-A; -B; -C; -DRB1 and -DQB1 loci using sequencing-based typing. Journal of the National Science Foundation of Sri Lanka, 47(3), pp.341–348. DOI: http://doi.org/10.4038/jnsfsr.v47i3.9292
Published on 30 Sep 2019.
Peer Reviewed

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