A simple and reliable assay for detecting specific nucleotide sequences in plants using optical thin-film biosensor chips.
Here we report the adaptation and optimization of an efficient, accurate and inexpensive assay that employs custom-designed silicon-based optical thin-film biosensor chips to detect unique transgenes in genetically modified (GM) crops and SNP markers in model plant genomes. Briefly, aldehyde-attached sequence-specific single-stranded oligonucleotide probes are arrayed and covalently attached to a hydrazine-derivatized biosensor chip surface. Unique DNA sequences (or genes) are detected by hybridizing biotinylated PCR amplicons of the DNA sequences to probes on the chip surface. In the SNP assay, target sequences (PCR amplicons) are hybridized in the presence of a mixture of biotinylated detector probes and a thermostable DNA ligase. Only perfect matches between the probe and target sequences, but not those with even a single nucleotide mismatch, can be covalently fixed on the chip surface. In both cases, the presence of specific target sequences is signified by a color change on the chip surface (gold to blue/purple) after brief incubation with an anti-biotin IgG horseradish peroxidase (HRP) to generate a precipitable product from an HRP substrate. Highly sensitive and accurate identification of PCR targets can be completed within 30 min. This assay is extremely robust, exhibits high sensitivity and specificity, and is flexible from low to high throughput and very economical. This technology can be customized for any nucleotide sequence-based identification assay and widely applied in crop breeding, trait mapping, and other work requiring positive detection of specific nucleotide sequences.
Bai
Su-Lan
SL
Peking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, and The National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China.
Zhong
Xiaobo
X
Ma
Ligeng
L
Zheng
Wenjie
W
Fan
Liu-Min
LM
Wei
Ning
N
Deng
Xing Wang
XW
D000483:Alleles; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D018744:DNA, Plant / Q000032:analysis / Q000235:genetics*; D017343:Genes, Plant / Q000235:genetics; D008957:Models, Genetic; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results
D018744:DNA, Plant; D015345:Oligonucleotide Probes; D010940:Plant Proteins