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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.