Fast Detection with High Sensitivity and Selectivity
Fast hybridization kinetics is one of the key features of DNA microarrays fabricated on NB slides. Both sides of the NB slides are coated with trillions of NanoCones™, cone-shaped organic chemical compounds. The apexes of NanoCones are functionalized with a NHS (N-hydroxyl succinimidyl) or aldehyde groups for attaching probe molecules with amino groups. These NanoCones can minimize steric hindrance and electrostatic disturbance by regularly controlling lateral spacing between surface-immobilized molecules, and eventually enhance binding kinetics between probe and target molecules.
The NB9 NHS slide was compared with two market leading slides for a DNA microarray assay to detect single base mutations of hot spots of p53 tumor suppress gene. The result demonstrated that the NB9 NHS slide showed up to 10 times enhanced sensitivity and up to 400% improved selectivity than other slides. In addition to this superiority, the NB9 NHS slide allows much faster detection than others. Even 10 min hybridization time in this study is sufficient to detect target DNAs with good reliability.
In this study a plasmid DNA including exon 5, 6, 7 and 8 of p53 gene was extracted from E. coli., and 32 ng of the plasmid DNA was labeled with a fluorescence dye during a random priming method. The random priming is known to increase the amount of target DNA 10 to 100 times in spite of much lower amplification yield than a PCR method. This labeled target DNA was used for hybridization on DNA microarrays. Hybridization was performed with a MAUI hybridization machine (BioMicro). The fluorescence images were acquired by a confocal laser scanner, ScanArray Lite (GSI Lumonics) and each fluorescence signal was analyzed by Imagene 4.0 software (Biodiscovery). For fair comparison, all processes were performed by following the protocols that the companies recommended in their user guides, and each company’s printing buffer was utilized.
Figure 1. A scheme showing selective DNA detection on the NB slide
While a DNA microarray manufactured on the NB slide provides each probe DNA with ample space for hybridization with incoming target DNAs, the microarray shows enhanced discrimination efficiency for various types of single nucleotide polymorphism (SNP). In model system, the highest discrimination efficiency holds for all tested cases (100:<1 for internal mismatched cases; 100:<28 for terminal mismatched cases). On the other hand, a competitor slide without controlled spacing shows relatively poor discrimination efficiency and for one terminal mismatched case, its signal is almost indistinguishable with that of the matched one. Here, capability to discriminate even the most challenging terminal mismatched case is demonstrated for the DNA microarray on the NB slide.
– Real system : Detection of 7 hot spots of p53 gene
Figure 2. Fluorescence images after the hybridization on the dendron-modified substrates. Single point mutations of seven codons of p53 gene were detected with 200-400 mer target DNA samples.
Table 1. Relative fluorescence intensity of seven hotspot codons in p53 gene on the NB slide .
The fluorescence intensity of perfect matched sequence of each codon was set to 100.
Gene expression profiling
DNA microarrays have made a dramatic impact on gene expression profiling studies. However, this format has not been standardized until now and is still suffering from low sensitivity for RNA (or cDNA) and undesirable variation within a microarray. Therefore, the NB slide is expected to provide a gold standard DNA microarray with the highest sensitivity and reliability for gene expression profiling.
For comparison with one of the most popular competing slides, gene expression profiling studies were performed using cDNAs reverse-transcripted out of total RNA without further amplification process. As shown in the figure below, NB slide shows very high sensitivity even using 1 μg total RNA, while the competitor has many missing spots as well as lower signal intensity than the NB slide. Moreover, the NB slide maintains a very low background signal for 1 μg total RNA without any blocking agents such as BSA, but the competitor has higher background signal in spite of being treated with BSA.
Figure 3. Fluorescence images after hybridization with cDNAs reverse-transcripted from total RNA. NB and competitor slides were tested.