Cost-effective TA cloning applied to Sanger sequencing and HLA allele typing

Anupama C Neduvat, Prerana M Murthy, Sudarson Sundarrajan, Sriram Padmanabhan

Abstract


The polymerase chain reaction (PCR)-based technology for clinical HLA typing involves DNA Sanger sequencing of the PCR amplified products of polymorphic loci of HLA such as HLA-A, -B, -C, -DPB1,-DQB1 and -DRB1 in support of the unrelated donor hematopoietic stem cell transplantation. The TA cloning of all the amplicons followed by Sanger Sequencing provides a primary screening tool of potential organ donors or to identify individuals who might have potential adverse drug responses. The Class I exon 2 and 3 genes were amplified as a single fragment and cloned into a TA cloning vector pTZ57R/T while the Class II exon 2 and 3 fragments were added together in a ligation mix to a TA cloning vector pTZ57R/T. Positive clones were subjected to Sanger sequencing and HLA alleles determined using IMGT database. Results indicate that the one could successfully clone all the exons of the HLA genes by the strategies described and hence could reduce the cost of TA cloning substantially. Furthermore, we were also successful in achieving amplification of all the desired amplicons as a multiplex PCR reducing the cost of the modified method further by nearly 60%. We present a cost-effective TA cloning strategy for achieving accurate allele typing of HLA at 2- digit resolution. Taken together, an efficient cloning methodology with a significant lower cost for accurate HLA typing presented here is encouraging. The data suggests that it may be employed for routine cloning of variable targets in molecular biology applications.


Keywords


Allele typing, cloning, HLA typing, PCR products, pTZ57R/T

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