Correlation of SHOX2 gene amplification and DNA methylation in lung cancer tumors.

BACKGROUND: DNA methylation in the SHOX2 locus was previously used to reliably detect lung cancer in a group of critical controls, including 'cytologically negative' samples with no visible tumor cell content, at a high specificity based on the analysis of bronchial lavage samples. This study aimed to investigate, if the methylation correlates with SHOX2 gene expression and/or copy number alterations. An amplification of the SHOX2 gene locus together with the observed tumor-specific hypermethylation might explain the good performance of this marker in bronchial lavage samples. METHODS: SHOX2 expression, gene copy number and DNA methylation were determined in lung tumor tissues and matched morphologically normal adjacent tissues (NAT) from 55 lung cancer patients. Quantitative HeavyMethyl (HM) real-time PCR was used to detect SHOX2 DNA methylation levels. SHOX2 expression was assayed with quantitative real-time PCR, and copy numbers alterations were measured with conventional real-time PCR and array CGH. RESULTS: A hypermethylation of the SHOX2 locus in tumor tissue as compared to the matched NAT from the same patient was detected in 96% of tumors from a group of 55 lung cancer patients. This correlated highly significantly with the frequent occurrence of copy number amplification (p < 0.0001), while the expression of the SHOX2 gene showed no difference. CONCLUSIONS: Frequent gene amplification correlated with hypermethylation of the SHOX2 gene locus. This concerted effect qualifies SHOX2 DNA methylation as a biomarker for lung cancer diagnosis, especially when sensitive detection is needed, i.e. in bronchial lavage or blood samples.

Immobilization of oligonucleotides with homo-oligomer tails onto amine-functionalized solid substrates and the effects on hybridization.

Microarrays have become important tools for the detection and analysis of nucleic acid sequences. Photochemical (254 nm UV) DNA immobilization onto amine-functionalized substrates is often used in microarray fabrication and Southern blots, although details of this process and their effects on DNA functionality are not well understood. By using Cy5-labeled model oligonucleotides for UV immobilization and Cy3-labeled complementary sequences for hybridization, we measured independently the number of immobilized and hybridized oligonucleotides on the microarray surface. By using a two-color fluorescence LED setup and a novel method to compile the data, a full analysis has been made of the effects of oligonucleotide composition (length and sequence) on both immobilization and hybridization. Short homo-oligomer sequences (tails) of uracils, thymines, and, to a limited extent, guanines attached to a hybridization sequence improve immobilization. We propose a possible mechanism explaining the grafting of these nucleotides to amine-functionalized substrates, and we found evidence that the DNA backbone is possibly involved in the immobilization process. Hybridization, on the other hand, greatly improves as a function of tail length regardless of tail composition. On the basis of statistical arguments, the probes increasingly bind via their tail, with the hybridization sequence becoming more accessible to its complement. We conclude that all tails, sequence independent, improve hybridization signals, which is caused by either improved immobilization (especially thymine and uracil) or improved hybridization (most pronounced with guanine tails).

Positive microcontact printing with mercaptoalkyloligo(ethylene glycol)s.

The soft lithographic replication of patterns with a low filling ratio by microcontact printing (microCP) is problematic due to the poor mechanical stability of common elastomeric stamps. A recently described strategy to avoid this problem employs a modified patterning method, positive microcontact printing ((+)microCP), in which a stamp with a mechanically more stable inverted relief pattern is used. In contrast to conventional negative microCP ((-)microCP), in the contact areas a self-assembled monolayer (SAM) is printed of a "positive ink", which provides only minor etch protection, whereas the noncontacted areas are subsequently covered with a different, etch-resistant SAM, prior to development by chemical etching. With the aim to identify novel, highly versatile positive inks, the patterning of gold by (+)microCP with mercaptoalkyloligo(ethylene glycol)s (MAOEGs), the subsequent adsorption of octadecanethiol (ODT), and the final development by wet chemical etching have now been studied. A polydisperse mixture of mercaptoundecylocta(ethylene glycol) derivatives was found to provide the best patterning results. The surface spreading of the positive ink during stamping, the exchange of printed MAOEGs with ODT, and the choice of the right etching bath were identified as key parameters that influence the achievable pattern resolution and contrast. Due to the modular composition of functionalized alkyloligo(ethylene glycol) derivatives, (+)microCP with these positive inks has the potential for easy adaptation to a variety of materials and development conditions.