Fabrication and optimization of HLA-DRB1-12 oligonucleotide microarray.
Oligonucleotide microarray is developed on the basis of hybridization on the solid substrate. The pre-activated glass substrates and the terminal modification of the oligonucleotides are the two important factors in the process of fabrication for microarray. In order to compare the hybridization signal intensity of the different terminal modified oligonucleotide probes, the eight kinds of oligonucleotides were designed according to the sequence of HLA-DRB1-12, including the amino modified oligonucleotides with PEG spacer and the one without spacer, the phosphorothioate modified oligonucleotides with PEG spacer and the one without spacer. They were modified on 5' terminal and 3' terminal, respectively. In addition, the oligonucleotides probes with the internal spacer of different number of PEG were designed to observe the relationship between the spacer of PEG and the hybridization efficiency. These probes were respectively fixed on the bromoacetylation activated and glutaraldehyde activated slides to manufacture the two kinds of microarray which hybridized with the fluorescence labeled PCR product of HLA-DRB1-12 gene. The results from the study demonstrated that the signal intensity of 3' amino-modified probes with the internal spacer of different number of PEG on the bromoacetylation activated slides was stronger than the others. It is concluded that the 3' amino-modified oligonucleotide with an internal PEG spacer and the bromoacetylation activated slide enhanced the hybridization efficiency and were worthy to be proposed for the fabrication of HLA microarray or other kinds of microarrays for detecting fluorescence labeled PCR product in the future study.
Li
Shuang-Ding
SD
The State Key Laboratory of Forensic Sciences of Xi'an Jiaotong University, Xi'an, 710061, China.
Tong
Li
L
Cheng
Su-Hong
SH
Ding
Yu
Y
Li
Sheng-Bin
SB
Wang
Sheng-Qi
SQ
D006684:HLA-DR Antigens / Q000235:genetics*; D059811:HLA-DRB1 Chains; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*
D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains