After elongation reaction an ion exchange resin (Spectro-CLEAN, Sequenom, Hamburg, Germany) was added to avoid adsorption/attachment of cations. Nucleic acids show a high affinity to alkali and alkaline earth ions. These cations disturb the MALDI TOF MS-analysis. The emerging single signals, which are attached to the actual molecule ion, cause a reduction of the signal-noise-ratio. Purification was carried out according to the standard protocol from Sequenom (Manual Processing Procedure fort he MassARRAY II System, Sequenom Hamburg, Germany).
iPLEX-products were spotted onto a 384 silicon chip by means of a nanoliter spotter. The chip is covered with a crystalline (3-Hydroxypicolinacid). Besides the 384 matrix spots, onto which the samples are transferred, 10 additional matrix spots are located on the chip. Onto these chips a calibrant (Sequenom Hamburg, Germany) spotted, containing a mixtrue of 3 oligonucleotides of known masses and serves for calibration of the analyzing system.
Fig. 5.: Schematic of iPLEX reaction
Depending on the present allele, products differing in length and mass are generated. These differences are subsequently shown by MALDI TOF mass spectrometry
2.10
SNP Detection by Means of Matrix-assisted Laser Desorption
Ionisation (MALDI) Time of Flight (TOF) Mass Spectrometry (MS)
MALDI TOF MS is an analytical method for determination of molecular masses of free ions in high vacuum. The MALDI TOF MS was developed 1987 by Hillenkamp und Karas. Formerly, this method was for mass determination of bigger molecules (such as peptides and proteins). Nowadays it is also used in SNP analysis.For sample preparation different techniques were developed, all guaranteeing intercalation of the analyte molecule into the lattice of the matrix (Karas and Hillenkamp 1988); (Kirpekar, Nordhoff et al. 1998). A common matrix is 3-hydroxy-picolin-acid. This is an aromatic, carboxylcontaining acid, giving its proton to the negative oligonucleotides of the sample in a lattice structure and therefore allowing ionisation. Matrix substance absorbs radiated laser energy and protects, due to the 100- to 1000-times overplus, the analyte molecules from its degradation. Moreover the matrix is to avoid damaging of the analyte and interaction of analyte molecules among each other and between analyte and sample carrier (Hillenkamp, Karas et al. 1991). Analyte and matrix build a so-called MALDI-plum.
During MALDI TOF MS the sample is irradiated few nanoseconds with short waved laser light. This yields a local breakup of the solid surface. The absorbed energy is passed down to the samples molecule, imbedded in the matrix. Through this the sample molecule is desorbed, ionized and vaporized. The whole process is carried out in a vacuum (Gut 2001); (Hillenkamp, Karas et al. 1991).
A negative electrode, located next to the sample, generates an electrostatic field. Positive sample ions are accelerated from the sample surface towards the analyzer. For MALDI-analyses a time of flight spectrometer is used. Mass determination is accomplished by exact measurement of elapsed time between start of ion from the sample until arrival at the detector. Ions with smaller masses and equal kinetic energy can be accelerated more than heavier ions. At constant acceleration voltage and flight distance the measured time of flight correlates with a certain mass (Griffin and Smith 2000).
Calibration is accomplished with a reference substance of known mass. Typical flight times at the MALDI TOF MS are around 100 microseconds and drift distance is 1 – 4 m (Fig. 6).
Fig. 6.: Schematic of MALDI TOF mass spectrometry according to Griffin TJ et al.
Vaporization of matrix by means of the laser the oligonucleotide is accelerated and moves in the field-free flight tube with mass- and chargedependent velocity towards the detector. Here dissociation and detection of two oligonucleotides with different masses/charges are described.
Thanks to the development of MALDI TOF mass spectrometry it is now possible, to determine DNA fragments in the range of 1000 to 9000 Da, i.g. 3 – 30 bases with an accuracy of 0.1 to 0.01 %. Therefore a mass range is opened up, where SNP analysis via primer extension products can be performed. Adequate reaction conditions and primers were calculated by the SpectroDesigner Software (see § 3.2.3) and the respective allele of the selected SNPs were analyzed by means of MALDI TOF MS (Fig. 7).
Fig. 7.: MALDI TOF MS analysis of the primer extension reaction product
MALDI TOF MS analysis of the primer extension reaction product of an example SNP [C/T]. A: shows the homozygot condition of the allele T, B: shows homozygot condition of the allele C and C: shows the heterozygot condition. (X axis – mass, Y axis – intensity)
Genotyping was performed simultaneously for several SNPs (multiplex reaction) in a 384-well format according to an optimized protocol. Primers were designed by the SpectroDesigner Software such that the received products can be divided by mass (Fig. 8.). Therewith, overlaps of different allelespecific products with the same mass were avoided. Another problem is the possible weak signal intensity of individual products.
Fig. 8.: MALDI TOF MS analysis of a multiplex reaction
MALDI TOF MS analyis of a multiplex reaction of seven genotyped biallelic SNPs: The mass spectrum shows the analysis of the primer extension reaction for seven example SNPs, example SNP 1 [G/A] (marked red), example SNP 2 [G/A] (marked in green), example SNP 3 [C/T] (marked in blue), example SNP 4 [G/A] (marked in light blue), example SNP 5 [G/A] (marked in dark red), example SNP 6 [G/A] (marked in light green) und example SNP 7 [G/A] (marked in pink). Asterisks show the mass region, where the respective primers without extension were detected.
Fig. 9.: MALDI TOF MS analyses of extension primers for APP
MALDI TOF MS analyses of example extension primers for APP. The x-axis presents the mass, the y-axis the intensity of the signal. Peaks for the same SNP are marked in the same colours.