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D P Little

Publications and source records attributed to D P Little.

At least 19 recordsLinked to original sources
PubMed2001

High-throughput development and characterization of a genomewide collection of gene-based single nucleotide polymorphism markers by chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We describe here a system for the rapid identification, assay development, and characterization of gene-based single nucleotide polymorphisms (SNPs). This system couples informatics tools that mine candidate SNPs from public expressed sequence tag resources and automatically designs assay reagents with detection by a chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry platform. As a proof of concept of this system, a genomewide collection of reagents for 9,115 gene-based SNP genetic markers was rapidly developed and validated. These data provide preliminary insights into patterns of polymorphism in a genomewide collection of gene-based polymorphisms.

Alleles↗
PubMed2001

Estimation of DNA sequence diversity in bovine cytokine genes.

DNA sequence variation provides the fundamental material for improving livestock through selection. In cattle, single nucleotide polymorphisms and small insertions/deletions (collectively referred to here as SNPs) have been identified in cytokine genes and scored in a reference population to determine linkage map positions. The aim of the present study was twofold: first, to estimate the SNP frequency in a reference population of beef cattle, and second, to determine cytokine haplotypes in a group of sires from commercial populations. Forty-five SNP markers in DNA segments from nine cytokine gene loci were analyzed in 26 reference parents. Comparison of all 52 haploid genomes at each PCR amplicon locus revealed an average of one SNP per 143 bp of sequence, whereas comparison of any two chromosomes identified heterozygous sites, on average, every 443 bp. The combination of these 45 SNP genotypes was sufficient to uniquely identify each of the 26 animals. The average number of haplotype alleles (4.4) per PCR amplicon (688 bp) and the percentage heterozygosity among founding parents (50%) were similar to those for microsatellite markers in the same population. For 49 sires from seven common breeds of beef cattle, SNP genotypes (1,225 total) were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) at three amplicon loci. All three of the amplicon haplotypes were correctly deduced for each sire without the use of parent or progeny genotypes. The latter allows a wide range of genetic studies in commercial populations of cattle where genotypic information from relatives may not be available.

Alleles↗
PubMed1998

Sequencing exons 5 to 8 of the p53 gene by MALDI-TOF mass spectrometry.

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.

Base Sequence↗
PubMed1997

Improved analysis of microsatellites using mass spectrometry.

The primer oligo base extension reaction combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, recently introduced by our group for detection of single-point mutations and small insertions/deletions, has been applied to the reliable quantification of nucleotide repeat units in microsatellites. The AluVpA DNA marker within intron 5 of the interferon-alpha receptor gene was chosen as the model system. By varying the dNTP/ddNTP mixtures used, the assay could also be directed to detect the location of second-site mutations within the repeats, resulting in identification of alleles not detectable by electrophoretic sizing methods and thus an increase of the polymorphism information content for a sampling of 28 unrelated individuals. The method results in highly informative mass signals and has the potential to increase the polymorphism information content for systems containing second-site mutations; thus it is a very attractive alternative technique in statistics-based gene mapping, cancer diagnostics, and forensic applications.

Base Sequence↗
PubMed1997

Detection of RET proto-oncogene codon 634 mutations using mass spectrometry.

Mutations located in the RET proto-oncogene at codon 634 associated with multiple endocrine neoplasia type 2A and medullary thyroid carcinoma are detected by low-resolution and high-resolution mass spectrometry schemes not requiring labeling or electrophoretic separation of diagnostic products. The former requires measurement by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of 21- to 27-mer oligonucleotides generated by a primer oligo base extension reaction. The latter is based upon direct measurement of artificial products which include the mutation site using matrix-assisted laser desorption ionization Fourier transform mass spectrometry. In this feasibility study a synthetic 25-mer representing the wildtype allele (7660.3 Da) was easily distinguished from G to A (7644.3 Da) and G to T (7635.3 Da) mutant alleles; the mutant alleles, which differed in mass by only 9.0 Da, were easily resolved when analyzed as a mixture. The results of both detection schemes were highly accurate and reliable, indicating mass spectrometry to be a high-quality alternative for future DNA diagnostics performed in clinical laboratories and genetic profiling studies.

Base Sequence↗
PubMed1997

Competitive oligonucleotide single-base extension combined with mass spectrometric detection for mutation screening.

A rapid, robust and widely applicable mutation detection scheme not requiring radioactivity or gel-based detection is introduced. It is a single-tube, competitive oligonucleotide single-base extension (COSBE) reaction using a pair of primers with the 3'-terminal base complementary to either the normal or mutant allele. Upon hybridization and addition of a polymerase and the nucleotide triphosphate corresponding to the next base after the primer, only those primers properly annealed (i.e., no 3'-terminal mismatch) are extended; products are resolved by molecular weight shifts as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (single-scan spectrum acquisition < < 1 s). For the cystic fibrosis delta F508 polymorphism, 28-mer "normal" (N) and 30-mer "mutant" (M) primers generate 29-mer (N + I) or 31-mer (M + 1) products for homozygotes and both for heterozygotes. Since primer and product molecular weights are relatively low (< 10 kDa) and the mass difference between these are at least that of a single approximately 300-Da nucleotide unit, a low-resolution mass spectrometer is suitable for such measurements.

Alleles↗
PubMed1997

Detecting CFTR gene mutations by using primer oligo base extension and mass spectrometry.

A new method for the reliable identification of localized variations in DNA by detection of associated diagnostic products with matrix-assisted laser desorption ionization time-of-flight mass spectrometry is described. The diagnostic products are generated by the primer oligo base extension (PROBE) reaction, which requires a single detection primer complementary to a region down-stream of a target strand's variable site. On addition of a polymerase, three dNTPs, and the fourth nucleotide in dideoxy form, the primer is extended through the mutation region until the first ddNTP is incorporated; the mass of the extension products determines the composition of the variable site. Tests for five cystic fibrosis mutations, including two exon 11 sites measured in a biplex reaction, and for differentiating between three common alleles of the poly(T) tract at the intron 8 splice acceptor site of the CFTR gene are presented. All experimental steps required for PROBE are amenable to the high degree of automation desirable for a high-through-put diagnostic setting. Furthermore, it requires no fluorescent, chemiluminescent, or radioactive labeling; the mass signals measured offer a far more analytically definitive signal, leading in all cases to high-quality unambiguous and easily interpreted results.

Alleles↗
PubMed1997

Identification of apolipoprotein E polymorphisms using temperature cycled primer oligo base extension and mass spectrometry.

The isothermal Primer Oligo Base Extension (PROBE) reaction combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for diagnostic product detection as recently introduced by our group is modified to incorporate temperature cycling during the primer extension step, resulting in enhanced levels of diagnostic product generation. Utilizing temperature cycled PROBE, the identities of two apolipoprotein E polymorphisms (codons 112 and 158) for differentiation of epsilon 2/epsilon 3, epsilon 3/epsilon 3, epsilon 3/epsilon 4, and epsilon 4/epsilon 4 genotypes were simultaneously determined. Primers specific for each site are extended by a series of bases unique to the identity of that variable site, producing low mass diagnostic products (M(r) < 9000) highly amenable to detection by mass spectrometry. The temperature cycled PROBE method has yielded unambiguous and correct diagnoses for all samples tested thus far. The increased amount of diagnostic product generated per primer by the cycling method makes possible faster spectrum acquisition due to the increased signal intensity, critical for future automated measurement of such samples.

Apolipoproteins E↗
PubMed1996

High-resolution MALDI Fourier transform mass spectrometry of oligonucleotides.

The matrix-assisted laser desorption/ionization (MALDI) method has been used with an external ion source Fourier transform mass spectrometer (FIMS) to analyze single-stranded, mixed-base oligomers of DNA. It is demonstrated that ultrahigh mass resolution (830 000 fwhm) can be achieved for small oligomers, and high resolution (136 000 fwhm) can be achieved for a 25-mer at m/z 7634. MALDI-FTMS can clearly separate the molecular ion peaks from analyte-matrix adduct peaks and alkali metal-containing species that result from replacement of hydrogen ions with sodium or potassium ions at multiple sites along the phosphate backbone. Previous MALDI-FTMS studies of oligonucleotides had two limitations: (1) low sensitivity due to difficulty in trapping the high kinetic energy ions made by the laser and (2) fragmentation of the ions due to the long delay (tens to hundreds of milliseconds) between their formation and detection. Both of these problems are alleviated in the present study. With the external ion source FTMS instrument, ions made by MALDI are injected at low energy into the analyzer cell by a rf-only quadrupole ion guide, captured by gating the voltage on the trapping plates, and cooled by a 0.5-s pulse of argon gas. Under these conditions, fragmentation is minimized, and DNA ions can be trapped in the FTMS analyzer cell for greater than 50 s. Sensitivity is also improved, as demonstrated by detection of 1 pmol of a single-stranded, mixed-base 20-mer of DNA, with a signal-to-noise ratio greater than 20:1.

Base Sequence↗
PubMed1996

Isotopic assignment in large-molecule mass spectra by fragmentation of a selected isotopic peak.

For large ( > 5 kDa) ionic species, Fourier transform ion cyclotron resonance instruments yield by far the highest mass accuracy. However, this can be compromised by misassignment of the isotopic content based on predicted natural abundances of the isotopic peaks. As an alternative method independent of natural abundance variations, high-resolution isolation and dissociation of a single isotopic peak yields a distribution of isotopic peak abundances characteristic of the isotopic content of the precursor peak. Accuracy is enhanced if the precursor peak is abundant and of minimum heavy isotope content, and if the product species is abundant and of intermediate mass. In addition, such spectra of the highest mass products are useful for identifying complementary product pairs, a key step in sequencing proteins and nucleotides.

Amino Acid Sequence↗
PubMed1996

Microfabrication and array technologies for DNA sequencing and diagnostics.

It is now possible to miniaturize numerous 'macroscale' processes and develop microfabricated devices to replace conventional equipment. Such advances have lead to the development of arrays of immobilized oligonuceotides useful for basic research, diagnostic studies, sequence analysis and a number of novel applications. Additionally, standard laboratory equipment has been redesigned on a microscale level to increase efficiency; many processes have been integrated onto one chip since miniaturization can be readily achieved.

Automation↗
PubMed1995

Direct sequence data from heterogeneous creatine kinase (43 kDa) by high-resolution tandem mass spectrometry.

Isoelectric focusing separation of recombinant rabbit muscle creatine kinase (CK) and its 282Cys-->282Ser mutant shows the presence of three and two isoforms, respectively, that exhibit equivalent enzymatic activity. Electrospray ionization coupled with Fourier-transform mass spectrometry (10(5) resolving power) of both CKs indicates that their major components are within +/- 2 Da of the M(r) value predicted from the cDNA sequences of these mixtures. Dissociation of (M + nH)n+ gives no evidence that the components of either CK are isomers; the masses of the 51 fragment ions correlate completely (+/- 1 Da) with the values predicted from the cDNA sequence and confirm the identities of 21 of the 380 amino acids and the 282Cys-->282Ser replacement in the mutant. The results are consistent with one or two steps of post-translational amidation/deamidation (NH2-->OH, 16 Da-->17 Da), each of which would produce only a 1 Da difference in M(r), with the fragment masses indicating that at least one modification occurs between residues 212 and 282.

Animals↗
PubMed1995

Attomole-sensitivity electrospray source for large-molecule mass spectrometry.

Full mass spectra of high resolving power are obtained from 0.2 nL sample volumes of large (> 10 kDa) nucleotides and proteins using a new electrospray ionization (ESI) system combined with Fourier transform mass spectrometry. The ESI needles are fabricated by laser-heated pulling of fused-silica tubing (5-20 microns i.d.), followed by chemical etching and surface metalization. Total analyte loaded at the instrument of 8.6 fmol and 216 amol produces signal-to-noise ratios of 400:1 and 60:1, respectively, and resolving power of > 10(5) for full mass spectra, while the total amount of material consumed is approximately 150 and 10 amol, respectively.

Animals↗
PubMed1995

Gas-phase folding and unfolding of cytochrome c cations.

Water is thought to play a dominant role in protein folding, yet gaseous multiply protonated proteins from which the water has been completely removed show hydrogen/deuterium (H/D) exchange behavior similar to that used to identify conformations in solution. Indicative of the gas-phase accessibility to D2O, multiply-charged (6+ to 17+) cytochrome c cations exchange at six (or more) distinct levels of 64 to 173 out of 198 exchangeable H atoms, with the 132 H level found at charge values 8+ to 17+. Infrared laser heating and fast collisions can apparently induce ions to unfold to exchange at a higher distinct level, while charge-stripping ions to lower charge values yields apparent folding as well as unfolding.

Animals↗
PubMed1995

Surface-induced dissociation of multiply-protonated proteins.

A novel surface design compatible with the open cell geometry allows nonglancing angle collisions of selected ions stored in a Fourier transform mass spectrometer. Dissociation efficiencies of 36%, 22%, and 14% are achieved for gramicidin S, melittin, and carbonic anhydrase (29 kDa), respectively. Ion neutralization by the surface, which is highly competitive for many singly-charged ions, is minimal, and dissociation products of hypervalent neutral species are not detected. Instead, the spectra are similar to those from collisionally activated and infrared multiphoton dissociation; the fragmentation pathways are relatively independent of the method of energy deposition. For carbonic anhydrase, however, the single event excitation inherent to surface-induced dissociation appears to minimize secondary fragmentation, a critical advantage for tandem mass spectrometry of such large ions. Electrically floating the open cell below ground greatly enhances the collection efficiency.

Carbonic Anhydrases↗
PubMed1995

Verification of 50- to 100-mer DNA and RNA sequences with high-resolution mass spectrometry.

Electrospray ionization with Fourier-transform mass spectrometry achieves accurate (< 50-ppm) determination of molecular weights of nucleotides, verifying structures of biological RNA and synthetic single-stranded DNA. High (1o(5)) resolving power makes possible detection of subpicomole impurities and adducts that confuse lower-resolution measurements. Molecular ions in a spectrum of 76-mer tRNA(Phe) had 34-55 Na adducts; when desalted, these show a molecular mass of 24,950.5 Da (expected, 24,950.3 Da) and minor variants at approximately -15 and +15 Da. A 50-mer DNA is characterized with < 10-ppm mass error, with detection of both N + 1 and N - 1 failure sequences. Special electrospray ionization conditions are necessary for a 72-mer to minimize fragmentation in the ion source. Despite the chemical noise from this, as well as failed sequences from automated synthesis, the spectrum of a 100-mer single-stranded DNA yielded a molecular mass of 30,702.4 +/- 1 Da, in good agreement with the expected value, 30,702.1 Da.

Base Sequence↗