Various 5'O-N-protected deoxynucleoside-3'-O-beta-cyanoethyl-N,N-dialkylamino-/N- morpholinophosphoramidites were prepared from beta-cyanoethyl monochlorophosphoramidites of N,N-dimethylamine, N,N-diisopropylamine and N-morpholine. These active deoxynucleoside phosphates have successfully been used for oligodeoxynucleotide synthesis on controlled pore glass as polymer support and are very suitable for automated DNA-synthesis due to their stability in solution. The intermediate dichloro-beta- cyanoethoxyphosphine can easily be prepared free from any PC1(3) contamination. The active monomers obtained from beta-cyanoethyl monochloro N,N- diisopropylaminophosphoramidites are favoured. Cleavage of the oligonucleotide chain from the polymer support, N-deacylation and deprotection of beta-cyanoethyl group from the phosphate triester moiety can be performed in one step with concentrated aqueous ammonia. Mixed oligodeoxynucleotides are characterized by the sequencing method of Maxam and Gilbert.

Silicon chips with immobilized target DNAs were used for accurate genotyping by mass spectrometry. Genomic DNAs were amplified with PCR, and the amplified products were covalently attached to chip wells via Nsuccinimidyl (4-iodoacetyl)aminobenzoate (SIAB) chemistry. Primer annealing, extension, and termination were performed on a 1-l scale directly in the chip wells in parallel. Diagnostic products thus generated were detected in situ by using matrixassisted laser desorption ionization mass spectrometry. This miniaturized method has the potential for accurate, highthroughput, low-cost identification of genetic variations.

A piezoelectric pipet is used to dispense arrays of low-nanoliter aliquots of matrix and DNA into individual etched wells on <1 in. silicon chips prior to their semiautomated analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Spectrum acquisition is expedited relative to conventional “large-spot” MALDI since the resulting miniaturized samples are approximately the same size as the irradiation area of the ionization/desorption laser; thus, searching for crystal regions from which intense analyte signals may be obtained is not necessary. Using a linear TOF instrument designed for scanning high-density arrays of samples, mass spectra from as little as 0.2 fmol (45 nM) of a 36-mer DNA have been acquired from single miniature elements. Spot-to-spot reproducibility from microdispensed samples is superior to that using conventional pipets; in less than 6 min, spectra with high signal-to-noise ratios were acquired from 100 elements containing 8 fmol of a 25-mer. Low-nanoliter quantities of DNA diagnostic products generated in primer oligo base extension reactions from PCR templates were transferred to chips and analyzed by MALDI-TOF MS, giving accurate genotyping results for single base mutations and short tandem repeat polymorphisms (microsatellites). These procedures provide enabling capabilities for extremely accurate high-throughput DNA diagnostics and sequencing based on mass spectrometry.

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DNA probes and synthetic oligonucleotides in general present one of the key tools in modern molecular biology research and increasingly also in commercial applications. Along with the many applications that have been developed for and with DNA probes, faster and more sensitive detection methods are being developed. One of the most promising recent developments presents a method based on enzymatically triggered chemiluminescence. Details of this chemistry along with applications in molecular biology and immunology will be discussed and compared to conventional methods.

A method is described for the covalent attachment of DNA to a solid surface at high density for hybridization detection by mass spectrometry. A silicon wafer is functionalized to place an amino group on the surface; a heterobifunctional cross-linking agent is then reacted with the primary amine to incorporate an iodoacetamido group. An oligodeoxynucleotide containing a 3'- or a 5'-disulfide is treated with a reducing agent, resulting in a terminal free thiol, which is then coupled to the iodoacetamido surface. Analysis of the surface reveals that the amount of covalently bound oligodeoxynucleotide is 250 fmol of DNA/mm(2) with ∼40% of the immobilized oligodeoxynucleotides available for hybridization. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF MS) analysis reveals that the covalent linkage to the support remains intact, only the annealed strand is desorbed by the laser, and the amount of DNA hybridized to the array is sufficient for detection.