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

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


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.


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


V Jung, SB Pestka and S Pestka. Cloning of polymerase chain reaction-generated DNA containing terminal restriction endonuclease recognition sites. Methods Enzymol.1993; 218, 357-62.

GL Costa and MP Weiner. Polishing with T4 or Pfu polymerase increases the efficiency of cloning of PCR fragments. Nucleic Acids Res. 1994; 22, 2423.

S Kaluz and AP Flint. Ligation-independent cloning of PCR products with primers containing non base residues. Nucleic Acids Res. 1994; 22 4845.

D Marchuk, M Drumm, A Saulino and FS Collins. Construction of T- vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 1991; 19, 1154.

MY Zhou and CE Gomez-Sanchez. Universal TA cloning. Curr. Issues Mol. Biol. 2000; 2, 1-7.

Y Zhao, Z Liu, S Yu, S Wen, L Hammarstrom and Rabbani H. Construction of a high efficiency PCR products cloning T vector using pGEM-5zf (+). Avicenna J Med Biotechnol. 2009; 1, 37–9.

JL Davies, Y Kawaguchi, ST Bennett, JB Copeman, HJ Cordell, LE Pritchard, PW Reed, SCL Gough, SC Jenkins, SM Palmer, KM Balfour, BR Rowe, M Farrall, AH Barnett, SC Bain and JA Todd. Genome-wide search for human type 1 diabetes susceptibility genes. Nature 1994; 371, 130–36.

TJ White, N Arnheim and HA Erlich. The polymerase chain reaction. Trends Genet. 1989; 5, 185–9.

FW Petersdorf and JA Hansen. A comprehensive approach for typing the alleles of the HLA-B locus by automated sequencing. Tissue Antigens 1995; 46, 73–85.

SGE Marsh, ED Albert, WF Bodmer, RE Bontrop, B Dupont, HA Erlich, M Fernández-Viña, D E Geraghty, R Holdsworth, C K Hurley, M Lau, K W Lee, B Mach, M Maiers, W R Mayr, C R Müller, P Parham, E W Petersdorf, T Sasazuki, J L Strominger, A Svejgaard, P I Terasaki, J M Tiercy, and J Trowsdale. Nomenclature for factors of the HLA system. Tissue Antigens 2010; 75, 291-455.

Y Bai, M Ni, B Cooper, Y Wei and W Fury. Inference of high resolution HLA types using genome-wide RNA or DNA sequencing reads. BMC Genomics 2014; 15, 325.

H Miyadera and K Tokunaga. Associations of human leukocyte antigens with autoimmune diseases:

challenges in identifying the mechanism. J Human Genet. 2015; 60, 697-702.

JM Blackwell, SE Jamieson and D Burgner. HLA and infectious diseases. Clin Microbiol Rev. 2009; 22, 370-85.

C Wang, S Krishnakumar, J Wilhelmy, F Babrzadeh, L Stepanyan, LF Su , D Levinsonc, MA Fernandez-Viñad, RW Davisa, MM Davise and M Mindrinosa. High-throughput, high-fidelity HLA genotyping with deep sequencing. Proc Natl Acad Sci. USA, 2012; 109, 8676–81.

SD Adams, KC Barracchini, D Chen, FM Robbins, L Wang, P Larsen, R Luhm and DF Stroncek. Ambiguous allele combinations in HLA Class I and Class II sequence-based typing: when precise nucleotide sequencing leads to imprecise allele identification. J. Transl. Med. 2004; 2, 30.

JJ Poh and SK Gan. Comparison of customized spin-column and salt-precipitation finger-prick blood DNA extraction. Biosci Rep. 2014; 34, e00145.

Y Itoh, N Mizuki, T Shimada, F Azuma, M Itakura, K Kashiwase, E Kikkawa, JK Kulski, M Satake and H Inoko. High-throughput DNA typing of HLA-A, -B, -C, and -DRB1 loci by a PCR–SSOP–Luminex method in the Japanese population. Immunogenetics 2005; 57, 717-29.

V Lange, I Böhme, J Hofmann, K Lang, J Sauter, B Schöne, P Paul, V Albrecht, JM Andreas, DM Baier, J Nething, U Ehninger, C Schwarzelt, J Pingel, G Ehninger, AH Schmidt. Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing. BMC Genomics 2014; 15, 63.

XQ Xing, YN Chu, Z Xiang, QX Song and GH Zhou. Establishment of cloning and sequencing method for high-resolution HLA-B genotype assay. Chin J Anal Chem 2014; 42, 1574–1579.

J Robinson, JA Halliwell, JD Hayhurst, P Flicek, P Parham P and SGE Marsh. The IPD and IMGT/HLA database: allele variant databases. Nucleic Acids Res. 2015; 43, 1-9.

J Robinson, MJ Waller, P Parham, N de Groot, R Bontrop, LJ Kennedy, Stoehr P and Marsh SGE. IMGT/HLA and IMGT/MHC: sequence databases for the study of the major histocompatibility complex. Nucleic Acids Res. 2003; 31, 311–14.

S Yao, DJ Hart and Y An. Recent advances in universal TA cloning methods for use in function studies. Protein Eng Des Sel. 2016; 29, 551-56.

M Palatinszky, M Nikolausz, D Sváb and K Márialigeti. Preferential ligation during TA-cloning of multi-template PCR products — a factor causing bias in microbial community structure analysis. J. Microbiol. Methods 2011; 85, 131–36.

SF Emonet, G Grard, NM Brisbarre, GN Moureau, S Temmam, RN Charrel and X de Lamballerie. Long PCR Product Sequencing (LoPPS): a shotgun-based approach to sequence long PCR products. Nat Protoc. 2007; 2, 340-46.

TA Yap, AD Smith, R Ferraldeschi, B Al-Lazikani, P Workman and JS de Bono. Drug discovery in advanced prostate cancer: translating biology into therapy. Nature Reviews Drug Discovery 2016; 15, 699–718.

N Cereb, P Maye, S Lee, Y Kong and SY Yang. Locus-specific amplification of HLA class I genes from genomic DNA: locus-specific sequences in the first and third introns of HLA-A, -B, and -C alleles. Tissue Antigens 1995; 45, 1-11.

C Lucan, LA Pop, A Florian, V Pileczki, B Petrushev, D Dima, I Frinc, I Berindan-Neagoe, A Irimie, C Berce, IS Florian, A Bojan and C Tomuleasa. HLA genotyping using next-generation sequencing. Rom. J. Intern. Med. 2016; 54, 98–104.

M Gowda, S Ambardar, N Dighe, A Manjunath, C Shankaralingu, P Hirannaiah, J Harting, S Ranade, L Jagannathan and Krishna S. Comparative analyses of low, medium and high-resolution HLA typing technologies for human populations. J Clin Cell Immunol. 2016; 7, 1-8.

S Boegel, M Lower, M Schafer, T Bukur, JD Graaf, V Boisguerin, Türeci O, Diken M, Castle JC and Sahin U. HLA typing from RNA-Seq sequence reads. Genome Med. 2012; 4, 102.

D Sengupta and B Cookson. SeqSharp: A general approach for improving cycle-sequencing that facilitates a robust one-step combined amplification and sequencing method. J Mol Diagn. 2010; 12: 272–77.

SJ Meltzer. Direct sequencing of polymerase chain reaction products. In: White BA (ed.). Methods in Molecular Biology. PCR protocols. Current methods and applications., Humana Press Inc. Totowa, NJ, 1993, p. 137-42.

CL Perng, LF Chang, WC Chien, TD Lee and JB Chang. Effectiveness and limitations of resolving HLA class I and class II by heterozygous ambiguity resolving primers (HARPs)--a modified technique of sequence-based typing (SBT). Clin Biochem. 2012; 45, 1471–78.

N Matamoros, M Moraru and C Vilches. Advancing allele group-specific amplification of the complete HLA-C gene—isolation of novel alleles from three allele groups (C*04, C*07 and C*08). Tissue Antigens 2013; 82, 280–85.


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