Design and Evaluation of Three Pair Primers for Exon 1 Amplification of Hyaluroglucosaminidase-1 Gene

Abstract

Problem statement: Hyaluronidase is an enzyme which catalyze hydrolysis of Hyaluronan (HA). Hyaluronan is important in cell migration during embryonic development, cellular proliferation and differentiation and has a structural role in connective tissues. Hyaluronidase deficiency is correlated with mucopolysaccharidosis IX. In human, hyaluronidase is encoded by HYAL1 gene. The mutation study of HYAL1 gene was carried out by many researchers, but until now, mutation study of HYAL1 still in progress and limited due to the lack of primer used in amplification of selected DNA sequence of HYAL1 gene and maximum length limitation imposed by DNA sequencer. Approach: The search for three pairs of primers for human exon 1 segmented amplification of HYAL1 gene was conducted and evaluated. The first step was to acquire HYAL1 gene sequence and then had it aligned with human chromosome 3 genomic contig sequence. Exon 1 of HYAL1 gene was found to be located at nt 201-1711 of the acquired human chromosome 3 genomic contig. Online Primer3 program was used to design three pairs of primers. The selected pairs of primer had been subjected to BLASTn operation for selectivity examination while onlineNetPrimer operation was carried out for examination of secondary structures. Results: The search for primers to amplify three different fragments of exon 1 of HYAL1 gene yielded three selected pairs of primers, namely forward primer 5’-TGACCCCCTACAAAAGCTCA-3’ (20 bp) and reverse primer 5’-AAGTCTCCGATTCCCCCACT-3’ (20 bp) for amplifying nt 1-551 of HYAL1 gene, forward primer 5’-AGTCCTGTGGGAGATGGCAGA-3’ (21 bp) and reverse primer 5’-CGGTAAATGTCCTTGGTGTCC-3’ (21 bp) for amplifying nt 355-1053 of HYAL1 gene and forward primer 5’-GCCATACCTGCTCCTGACTT-3’ (20 bp) and reverse primer 5’-ACAAGGTGGGCAGGTTACAG-3’ (20 bp) for amplifying nt 956-1511 of HYAL1 gene. When using these primers, nt 1-46 of amplified product of the first pair of primers and nt 555-584 of amplified product of the third pair of primers must not be considered because those are not part of exon 1 of HYAL1 gene. The results from both operations and trial to real samples using these primers indicated that all three pairs of primer were satisfactory for use. Conclusion/Recommendations: The three pairs of primers could be used to amplify specified segments of HYAL1 gene.