Application a Method of PCR Products Purification by Magnetic Beads Assay in HLA Sequence-Based Typing

Abstract

Objective To study and establish an effective method for purification of PCR products by the magnetic beads assay and apply the established method in human leukocyte antigen (HLA) sequencebased typing.Methods A fragment of approximate 2 000 bp target sequence of HLA-C gene was amplified covering from exon 1 to exon 4.PCR products of 96 samples were purified by OMEGA magnetic beads purification kit,and mixture of exonuclease I and shrimp alkaline phosphatase (SAP) (USB and Atria HLA-C SBT plus kit),respectively.Purified PCR products were subjected to sequence-based typing at exon 2,3 and 4 of HLA-C gene.Nucleotide sequencing was performed using an ABI 3730 DNA sequencer and allele alignments were conducted with Assign version 3.5.1.45 software (Conexio Genomics,Applecross,Australia).The average of BCS value (base calling score) for each sample was calculated.Results The mean of BCS value was 81.191 ± 3.539 using the OMEGA magnetic beads assay.The BCS value was 79.946±3.778 using the Atria ExoSAP-IT and the mean of BCS value was 70.384 ±5.778 using the USB mixture of exonuclease I and SAP purification method.The peak height for nucleotide A,G,C and T were all more than 1 000 RFU for all the 96 tested samples purified by OMEGA magnetic beads purification kit,mixture of exonuclease I and SAP (USB and Atria HLA-C SBT plus kit).The expense for purification of 10 μL PCR products using the magnetic beads purification kit was cheaper than using USB mixture of exonuclease I and SAP purification method.Conclusion Purification of PCR products using the magnetic beads assay has the advantage of inexpensive cost-consuming,and it doesn't need to be saved under frozen condition.It shows a broad foreground. Key words: human leukocyte antigen; sequence-based typing; purification of PCR products