Monica Brivio

Dried reagents for multiplex genotyping by tag-array minisequencing to be used in microfluidic devices.

We present an optimized procedure for freeze-drying and storing reagents for multiplex PCR followed by genotyping using a tag-array minisequencing assay with four color fluorescence detection which is suitable for microfluidic assay formats. A test panel was established for five cancer mutations in three codons (175, 248 and 273) of the tumor protein gene (TP53) and for 13 common single nucleotide polymorphisms (SNPs) in the TP53 gene. The activity of DNA polymerase was preserved for six months of storage after freeze-drying, and the half-life of activities of exonuclease I and shrimp alkaline phosphatase were estimated to 55 and 200 days, respectively. We conducted a systematic genotyping comparison using freeze-dried and liquid reagents. The accuracy of successful genotyping was 99.1% using freeze-dried reagents compared to liquid reagents. As a proof of concept, the genotyping protocol was carried out with freeze-dried reagents stored in reaction chambers fabricated by micromilling in a cyclic olefin copolymer substrate. The results reported in this study are a key step towards the development of an integrated microfluidic device for point-of-care DNA-based diagnostics.

A Total Integrated Biochip System for Detection of SNP in Cancer

Cancer is a leading cause of death worldwide. Resistance of tumor cells to radiation and chemotherapy is the major obstacle in cancer treatment. The serious toxicity that follows the administration of certain drugs can be associated with Single Nucleotide Polymorphisms (SNPs) of genes involved in drug metabolism and can ultimately reduce the clinical efficacy of chemotherapy. The KRAS and TP53 genes are altered in many human tumors. K-RAS point mutation appear early in the tumorigenesis pathway and can therefore be used for early cancer detection. The functional inactivation of p53 by point mutation is a hallmark of many tumors. Importantly, most anticancer agents act by inducing apoptosis and, typically, p53- deficient tumors are more resistant to drug-induced apoptosis than those carrying wild-type p53. It is therefore important to analyze such SNPs and point mutation in cancer diagnosis and treatment. In this paper, we described a total integrated BIOLABCHIP namely SMART-BioMEMS for rapid detection and identification SNPs of Kirsten-RAS (K-ras) and TP53 genes in cancer. The SMART-BioMEMS system consist of different components: An optical readout system, disposable biochip; and a reusable actuators chip that can perform all the steps from sample preparation, DNA isolation and purification, PCR amplification, Enzymatic clean up, mini-sequencing and SNP detection. The prototype of the SmartBIOMEMS system designs, detail of different components and functions will be present and discusses. It is results of great cooperation in the FP6 EU project SMART-BioMEMS.