PCR引物浓度对寡核苷酸液相杂交的影响

Effects of the primer concentration on the liquid-phase oligonucleotide hybridization

目的考察PCR引物浓度配比对液相微珠杂交效率的影响,寻求具有较强杂交信号和较好稳定性的PCR引物浓度配比。方法建立HLA-DRB1等位基因的相关数据库,选择在HLA-DRB1位点的第二外显子上设计探针,并且选择其保守序列作为阳性对照探针(DPC2),DPC2探针中间位点T突变成A作为阴性对照探针(DNC)。分别针对标本C2-008、C2-024、C2-025的等位基因序列设计出6条约21bp的寡核苷酸探针,各探针5’端用氨基(NH2)修饰。通过引物浓度梯度配比(1:100、1:50、1:20、1:8、1:4、1:2、1:1),对型别已知的细胞株DNA进行PCR扩增并得到目的片段(1:100配比除外),在相同条件下将PCR产物与寡核苷酸探针进行液相杂交检测。结果浓度配比为1:1的对称式扩增产物杂交结果不理想,而浓度配比分别为1:20、1:8、1:4、1:2的不对称扩增均得到了待检单链、双链DNA混合物,其中1:4浓度配比具有最好的扩增效率和稳定性。根据阳性信号与阳性标本是否相符表明:引物浓度配比为1:100的不对称PCR和1:1的对称PCR检测效果差,易出现假阴性;1:2、1:4、1:8、1:20配比检测效果较好,比较稳定。结论PCR引物浓度配比影响液相微珠杂交效率,不对称PCR产物有利于提高杂交效率,为快速成功配制PCR试剂奠定了基础,有利于寡核苷酸液相芯片的应用。

Objective This study investigated the impact of ratios of PCR primer concentration on the liquid-Bead hybridization efficiency to find appropriate ratio of primer concentration with a stronger hybridization signal and the hybrid stability.Methods Database of HLA-DRB1 alleles was established.Probes were designed according to the sequence of exon 2 of HLA-DRB1 locus.Conserved sequence were selected for positive control probe design（DPC2）.Negative control probe was synthesized through a mutation from T to A in the middle of the sequence of DPC2.Six oligo probes about 21 bp were designed specially targeted to allele sequences from samples C2-008,C2-024 and C2-025 separately.The 5＇-terminal sequences of each probe were modified by NH2.Genomic DNA from cell lines with known types was amplified with different ratios of primer concentration （1：100,1：50,1：20,1：8,1：4,1：2,1：1）.Target fragments were obtained except from amplification with 1：100 ratio.PCR products were hybridized with oligonucleotide probes by liquid hybridization under same conditions. Results Hybridization results from symmetric PCR products by concentration ratio of 1：1 are not satisfactory,while mixture of single-strand DNA and double-stranded DNA under test was obtained from asymmetric amplifications by ratios of 1：20,1：8,1：4,and 1：2,in which the best amplification efficiency and stability from the concentration ratio of 1：4.From the consistency of positive samples and positive signals,false negative results were common due to ratios of primer concentration 1：100 and 1：1. Conclusions The concentration ratio of PCR primers affects the efficiency of liquid-phase bead hybridization.Asymmetric PCR products help improve the hybridization efficiency.Our results established the foundation to prepare PCR reagents quickly and successfully in favour of liquid-phase oligonucleotide chip applications.Better results and stability would be expected using other ratios.