G. De Bellis

Time-resolved DNA-microarray reading by an intensified CCD for ultimate sensitivity.

We describe a novel technique for DNA-microarray reading based on time-resolved fluorescence measurements. We used an intensified CCD camera with picosecond resolution to acquire a set of time-delayed fluorescence images from a mutation DNA microarray marked with cyanine 3. We measured the fluorescence lifetimes of the marker and the background separately, and we used this information to calculate the amplitude map of the marker, starting from the time-delayed images. This procedure allowed us to identify hybridized spots that are not visible in fluorescence images acquired with continuous-wave detection.

Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data

Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need for positive controls or operator intervention to set signal thresholds when one allele is rare. In the interest of optimizing real-time PCR genotyping, we developed an algorithm for automatic genotype calling based on the full course of real-time PCR data. Best cycle genotyping algorithm (BCGA), written in the open source language R, is based on the assumptions that classification depends on the time (cycle) of amplification and that it is possible to identify a best discriminating cycle for each SNP assay. The algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare. Here, we describe the algorithm and test its validity, compared to the standard end-point method and to DNA sequencing.

Time resolved reading of DNA microarray by an intensified CCD camera

Summary form only. The DNA microarray technology is the most appealing tool for genetic investigation that has been devised in recent years. Microarrays basically consist of a matrix of thousands or even more spots of DNA fragments or oligonucleotides arranged on a microscope slide. If a probe, marked with fluorescent molecules, containing a base sequence complementary to one in the array is spread onto the slide, hybridization takes place and the hybridized spot can be easily located by fluorescent imaging. Presently, a laser scanner and a photomultiplier are used for DNA microarray reading and the discrimination between different markers is made on the basis of different absorption/emission wavelengths. In the present paper we introduce, for the first time to our knowledge, a technique to read DNA microarrays based on an intensified CCD camera having a picosecond resolution electronic shutter. This device permits to map the fluorescence intensity in a microarray in just one shot.