On-chip oligonucleotide ligation assay using one-dimensional microfluidic beads array for the detection of low-abundant DNA point mutations.
The detection of low-abundant DNA point mutations is very important for the early prediction of cancer, diagnostics of disease and clinical prognosis. In this paper, an on-chip oligonucleotide ligation approach that arrayed a series of functionalized beads in a single microfluidic channel was described for detection of low-abundant point mutations in p53 gene. This gene carried the point mutation with high diagnostic value for assessment of tumor progression and resectional borders. This work extended our prior efforts using one-dimensional (1-D) microfluidic beads array for protein and nucleic acid molecular profiling, and displayed high discrimination sensitivity to mutations detection due to the enhanced mass transport capability caused by microfluidic addressing format of beads array. As a demonstration, it was found that the on-chip beads ligation held high mutation discrimination sensitivity in 1 pM quantities at a SNR (signal-to-noise ratio) >2 using synthesized DNA oligonucleotides in accordance with target fragment. The RT-PCR products of tumor cell line A549, CNE2 and SKBr-3 were further examined to distinguish the point mutation at codon 175 of p53 gene. This approach was capable of detecting a point mutation in a p53 oncogene at a level of 1 mutant in 1000 wild-type sequences using PCR products without the need of LDR amplification. Additionally, this on-chip beads ligation approach also displayed other microfluidic-based advantages of simple handling (one sample injection per test), little reagent quantities, and low potential of contaminations.
Zhang
He
H
State Key Laboratory of Chemo/Biosensing & Chemometrics, Biomedical Engineering Center, College of Chemistry & Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha, PR China.
Yang
Xiaohai
X
Wang
Kemin
K
Tan
Weihong
W
Li
Huimin
H
Zuo
Xinbing
X
Wen
Jianhui
J
D004252:DNA Mutational Analysis / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity