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.
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.
Matrix-assisted laser desorption ionization time of flight mass spectrometry was used to sequence exons 5 to 8 of the human p53 gene. A single tube procedure was established for target amplification and mass spectrometric (MS) sequencing. The MS sequencing scheme is designed for high throughput and parallel sample processing, and is amenable to full automation. Reliable sequencing data were obtained using fmol sample amounts. The high resolution and accuracy of MS sequencing was demonstrated by direct sequencing of a heterozygous template.
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.