The ABI 3730xl DNA Analyzer, a 96-capillary sequencer, is the most robust sequencer on the market. With its production capacity of more than one million nucleotides per day, it is ideal for executing large sequencing projects. Brought to the market in 2002 by Applied Biosystems, we were the first in Canada to purchase one that same year. We conduct all of our sequencing projects on the ABI 3730xl.
In addition to its 96 capillaries, the ABI 3730xl is equipped with an electrophoresis system, a laser, and a CCD camera. The capillaries, having a diameter of approximately 250 um, are filled with a polymer that acts as a molecular sieve. The DNA molecules are introduced into one end of the capillaries by electroinjection, and migrate through these capillaries under the attraction of high voltage (8500 V) at which point the molecules are separated according to their lengths. Close to the anode, a laser passes through each capillary and excites the fluorescent molecules incorporated onto the DNA during the reaction sequence.
A CCD camera collects the information emitted by the fluorescent molecules as they pass in front of the laser. As each one of the four nucleic bases (A, C, G, and T) associates with a specific fluorochrome, the system distinguishes one base from another according to their respective wavelengths emitted. Subsequent data is analyzed, “base called,” and filed when electrophoresis is complete. We use the Sanger sequencing method, which takes advantage of the size separation of DNA molecules enabled by four fluorochrome-labeled ddNTPs.
The ABI 3130xl Genetic Analyzer, for all intents and purposes, is the same type of apparatus as the ABI 3730xl. The key difference resides in the number of capillaries, the ABI 3130xl being limited to 16. This was the first apparatus of its generation that Applied Biosystems put on the market. We mainly use it for multiplexed genotyping of microsatellite markers. This technique, which involves amplifying polymorphic regions with repetitive sequences(microsatellites) in the genome, generates alleles having variable fragment lengths. When one of the PCR primers is marked with a fluorochrome, the exact length of each of the studied microsatellites can be determined via capillary electrophoresis. The ABI 3130xl can detect 5 different fluorochromes, and each fluorochrome can be used more than once when there is a difference in length of more than 100 nucleotides between the fragments. Given that, we can multiplex up to 12 different markers per person using the same sample.
The Sequenom SpectroCHIP is a small, microarray chip that can furnish up to 384 SNP samples on its 384 individual pads. Exclusively-created for MALDI-TOF mass spectrometry, each SpectroCHIP is surfaced with silicon dioxide and holds prespotted matrices composed of 3-hydroxypicolinic acid (3-HPA). When purified nucleotide solution comes into contact with 3-HPA, a crystallization reaction takes place where the supernatant evaporates and only nucleotides remain. Shortly thereafter, those isolated SNP alleles’ masses can be measured on the mass spectrometer and data can be retrieved. Each SpectroCHIP has 10 calibrant wells to hold the special 3-points calibrant used as a standardizing tool for the mass spectrometer and a positive control.
MassARRAY Samsung Nanodispenser
The Samsung Nanodispenser are one of the pintool robots available from Sequenom Inc. It features a 24-pin head capable of aspirating DNA solution from 384-well microplates and dispensing 15 nl from each well onto 384-pad SpectroCHIPs. Apart from the 24-pin unit, the single-pin unit is used to dispense 3-points calibrant onto calibrant wells and can also be programmed to dispense samples from a microplate. All pins are sterilized before and after each dispense in a sonicator containing ethanol thus avoiding sample contamination. An internal camera is used as a tool to view an enlarged image of dispensed droplets before they crystallize onto the matrices. Prior to spotting an experimental chip, a user can run the visual inspection for droplet uniformity and adjust dispensing speeds accordingly to provide the best quality samples for mass spectrometer data acquisition. Typically, a complete 384-well plate can be dispensed onto 1 chip in less than 10 minutes.
Compact MALDI-TOF Mass Spectrometer
The Compact MALDI-TOF mass spectrometer provides a rapid way to obtain SNP genotype calls, spectra, and data quality scores in real-time. At high voltage, the laser precisely targets and fires at regions within each SpectoCHIP pad. The molecules are released to take a quick flight through the vacuum column. Since each molecule has a specific mass, each SNP allele’s mass can be determined based on its speed, or in other words, its time-of-flight. The RT software has the ability to take the best possible acquisition and assess that value to the final genotyping call. On the RT screen, the internal camera displays the real-time image of each pad as it is being shot by the laser. After data acquisition is completed, further analysis is accomplised on Typer software.
JANUS Automated Workstation
The Perkin Elmer JANUS Automated Workstation delivers many dispense technologies reunited on one instrument. These are very performing, effective and multi-purpose robots. They are been designed to automated the preparation and the manipulation of samples. Compatible with tubes, vials, 96-well microplates and 384-well microplates, they are adaptable to any application labware. Our three JANUS station, including one work-station located in a pre-PCR room, are equiped with an 8-tip dispense arm Varispan and two of these stations also have an 96-tip Modular Dispense Technology (MDT) dispense arms. These two arms are independant from each other and moves in the three cartesian directions X-Y-Z. The pipetting range starts at 2 uL and goes to 1 mL. Their flexibility offer us the automation of our protocoles and the reproductibility of our results.
Qubit Fluorometer 3.0
The Qubit Fluorometer 3.0 is the next-generation of the benchtop fluorometer who measure precisely the DNA, the RNA or the protein in your samples. The Qbit use dye who is selective for the biomolecule of interest and didn’t detect other molecule that maybe present in the sample. These dyes emit only when bound to the molecule, even at low concentration.