Application of DNA-binding polymers for preparation of DNA for analysis by matrix-assisted laser desorption/ionization mass spectrometry.

The susceptibility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to the presence of salts in a sample, especially salts of alkali metals, requires careful and often tedious desalting procedures which complicate and slow the throughput of MS-based methods. A novel approach to sample preparation was developed based on the extraction of DNA out of solution onto a solid surface with an attached DNA-binding polymer, such as polyethyleneimine or polyvinylpyrrolidone. The observed binding is strong enough to sustain washing, and, as a result, desalting and concentration can be performed in a single fast step. After DNA has been immobilized on the surface and supernatant solution removed, subsequent addition of MALDI matrix releases material from the surface, which co-crystallizes with matrix. The mass spectrometric analysis is then performed directly from this support. Analysis of oligonucleotides and three-fold multiplexed SNP typing reactions performed by this method shows improved sensitivity and excellent resolution for various DNA fragments, together with high tolerance to various buffer components, such as alkali metals and surfactants. Simplicity and speed make it attractive for high-throughput sample preparation and analysis of oligonucleotide mixtures by MALDI-MS.

Quantitative approach to single-nucleotide polymorphism analysis using MALDI-TOF mass spectrometry.

Pooling of DNA samples before genotyping is a valuable means of streamlining large-scale genotyping efforts in disease association studies, single-nucleotide polymorphism (SNP) validation or mutant allele screening programs. In this report, we explore the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to quantitative analysis of SNPs. The measurements are based on MALDI-TOF MS analysis of primer extension assays performed on standard mixtures of pooled PCR products at several test loci. The inherent high molecular weight resolution of MALDI-TOF MS conveys high specificity and good signal-to-noise ratio for performing accurate quantitation. The methods described maximize the sensitivity and quantitative capacity of MALDI-TOF MS while preserving the throughput and economic advantages of the MALDI-TOF platform. Using the format described, we demonstrate that allele frequencies as low as 5% can be detected quantitatively and unambiguously.

Multiplex genotyping of PCR products with MassTag-labeled primers.

A simple mass spectrometric based assay, the PinPoint assay, has previously been described for typing single nucleotide polymorphisms. The identity of a polymorphism is determined by mass differences of single base extended genotyping primers as determined by MALDI-TOF mass spectrometry. A simple method for multiplexing the assay is described, employing multiple primers with 5'oligo(dT) sequences (MassTags) which serve to mass discriminate the peaks of multiple extended and non-extended primers. The assay is extremely rapid and requires no labeling reagents.

Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry.

We report a simple method, the PinPoint assay, for detecting and identifying single-base variations (polymorphisms) at specific locations within DNA sequences. An oligonucleotide primer is annealed to the target DNA immediately upstream of the polymorphic site and is extended by a single base in the presence of all four dideoxynucleotide triphosphates and a thermostable DNA polymerase. The extension products are desalted, concentrated, and subjected to delayed-extraction MALDI-TOF mass spectrometry. The base at the polymorphic site is identified by the mass added onto the primer. Heterozygous targets produce two mass-resolved species that represent the addition of both bases complementary to those at the polymorphic site. The assay is suitable for double-stranded PCR products without purification or strand separation. More than one primer can be simultaneously extended and then mass-analyzed. The mass spectrometric method thus shows promise for high-volume diagnostic or genotyping applications.

Matrix-assisted laser desorption/ionization for sequencing single-stranded and double-stranded DNA.

The DNA sequence of a single-stranded and double-stranded template was determined. The templates were sequenced using the chain termination method and cycle sequencing method and detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The sequencing products were analyzed successfully without the laborious and expensive methods for removal of the template. Direct sequencing of the double-stranded template was achieved with minimal post-reaction purifications, which could be extremely important for mutation analysis and clinical diagnosis. A systematic study of the mechanisms and kinetics of sequencing reactions was also performed. The details of this analysis and directions for future improvements of the quality of sequencing are presented.

DNA sequence analysis by matrix-assisted laser desorption ionization MS.

The major advantage of this procedure is that, as detailed in Figure 5, the entire sequence of an oligonucleotide is determined in less than 1 h using inexpensive reagents. DE MS, because of its significantly higher resolving power, permits analysis of oligonucleotides as long as 50 bases, about twice as long as that which is possible without DE. Phosphorothioates can be sequenced after oxidation to the phosphodiester state. We have also found that the enzymes will digest through a number of modifying and protecting groups on DNA and RNA, including 2'-amino- and 2'-alkyl-substituted hydroxy groups. The resultant spectra can confirm not only the presence, but also the position, of modifying groups on both DNA and RNA molecules.

Sequencing oligonucleotides by exonuclease digestion and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A protocol was developed for sequencing oligonucleotides by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Oligonucleotides were partially hydrolyzed in separate time-course digestions with 3' --> 5' and 5' --> 3' acting phosphodiesterases in a MALDI-compatible buffer or in the MALDI matrix itself. The partial digests were analyzed by MALDI-TOF mass spectrometry employing an instrument equipped with delayed ion extraction and the sequence was inferred from the mass differences between adjacent peaks. Resolution, mass accuracy, and sensitivity were considerably enhanced with delayed extraction, in comparison with the standard MALDI technique. Much longer lengths of DNA can be unambiguously sequenced with delayed extraction MALDI compared with standard MALDI-MS.

DNA sequencing by delayed extraction-matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry was used to detect and order DNA fragments generated by Sanger dideoxy cycle sequencing. This was accomplished by improving the sensitivity and resolution of the MALDI method using a delayed ion extraction technique (DE-MALDI). The cycle sequencing chemistry was optimized to produce as much as 100 fmol of each specific dideoxy terminated fragment, generated from extension of a 13-base primer annealed on 40- and 50-base templates. Analysis of the resultant sequencing mixture by DE-MALDI identified the appropriate termination products. The technique provides a new non-gel-based method to sequence DNA which may ultimately have considerable speed advantages over traditional methodologies.

Applications of delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry to oligonucleotide analysis.

A delayed ion extraction technique is shown to dramatically improve mass resolution and the overall quality of matrix-assisted laser desorption ionization (MALDI) mass spectra of oligonucleotides. Isotope limited mass resolution was obtained on samples up to 10-kDa molecular mass in linear mode, and as high as 7500 mass resolution (defined at half peak height) was observed in reflector mode. This performance is as good as that achieved to date for peptides and proteins. Applications included the detection of oxidized byproducts of phosphorothioate DNA and separation of components differing only by 15 Da at 9.5-kDa molecular mass. In addition to single components, complex mixtures could also be analyzed at greatly improved performance over conventional MALDI. An example is shown for sequence verification of an oligonucleotide of 31 bases in length by analyzing the failure products. Mass accuracy was adequate to verify sequences of oligodeoxyribonucleotides up to 9500-Da molecular mass. Fast fragmentation taking place between the ionizing pulse and the extraction pulse is demonstrated to be a sequencing tool for small oligonucleotides. By proper selection of matrix material, wavelength, and irradiance, fast fragmentation can be promoted efficiently. Fragment ions tend to form from cleavage of phosphodiester bonds, as previously observed in infrared MALDI.

Improved quantitative PCR using nested primers.

Quantitative PCR can often be improved by conducting the amplification with nested primers. First, fewer nonspecific amplification products, which could otherwise interfere with quantitation, are produced. Often, nonspecific products can be eliminated. In these cases, relatively simple nonspecific detection techniques are suitable for quantitation. In addition, nested primer PCR provides intrinsic PCR product carryover protection and generally improves the robustness and lower limit of detection of PCR. For a nested PCR to provide useful quantitative information, it is important that the initial phase of amplification, performed with the outer pair of primers, takes place entirely in the exponential phase. This is generally achieved easily. The major consideration in designing a nested PCR protocol compatible with quantitation is to assure that the maximum concentration of PCR products produced by the outer primers does not exceed approximately 10% the molarity of the outer primers. A simple formula can be used to determine the maximum number of thermal cycles that provide this assurance. Good correspondence was obtained between initial target concentration and final PCR product yield in a nested-primer HIV-1 PCR.

Detection of bacterial mRNA using polymerase chain reaction.

A method was developed for the detection of bacterial mRNAs using reverse transcriptase followed by the polymerase chain reaction (PCR) and Southern blot analysis. The method involves brief inhibition of protein synthesis with chloramphenicol, followed by reverse transcription, PCR amplification of cDNA and Southern blot hybridization. Detection of mRNAs by reverse transcription-PCR-Southern blot analysis is orders of magnitude more sensitive than Northern blot hybridization.

Rapid separation, quantitation and purification of products of polymerase chain reaction by liquid chromatography.

The polymerase chain reaction (PCR), a new, powerful method for rapid enzymatic amplification of specific DNA fragments, has gained tremendous popularity in molecular biology. This paper describes the successful application of liquid chromatography to the analysis of products of the PCR. Efficient separation of both DNA restriction fragments and amplified PCR products were achieved in 10-12 min on a new ion-exchange column, DEAE-NPR, packed with 2.5-microns non-porous particles. The PCR products were quantitated with a reproducibility within 10%. Use of liquid chromatography was demonstrated for separation and quantitation of PCR products in amounts below those required for direct analysis by ethidium bromide gel electrophoresis or a Hoechst 33258 dye-based fluorescence assay. Liquid chromatography was also demonstrated to be effective for quick optimization of PCR procedures.

A new technique for desorbing substances tightly bound to affinity gels: flat-bed electrophoretic desorption in Sephadex via isoelectric focusing (FEDS-IEF).

In some cases, proteins and other molecules which are tightly bound to affinity gels can be recovered under mild conditions by electrophoresis. We have extended this technique by running electrophoretic desorption in flat-beds of Sephadex in the presence of ampholytes (FEDS-IEF). A number of advantages of this technique are noted: due to the geometry of the apparatus, high voltages can be used which result in short running times; there are no physical barriers to the migration of the protein and no abrupt conductivity drops; desorbed samples are easily located and recovered; and relatively large sample loads can be readily accommodated. Running times are very sensitive to the experimental conditions. Affinity gels should be applied as a narrow zone, distant from the anticipated banding position of the desorbed species. A wide ampholyte interval is generally recommended. The system appears to be gentle and flexible enough to allow investigators to optimize the conditions for desorption of various affinity gel systems.

Production of Ficoll, Percoll, and albumin gradients by the freeze-thaw method.

Ficoll, sucrose, albumin, and Percoll, a modified colloidal silica centrifugation medium, all form density gradients upon freezing and thawing. The data suggest that any density gradient material will form gradients upon freezing and thawing, provided the material is stable to freezing.

Fractionation of water-insoluble protein using sephacryl S-200 in formamide.

The ability of Sephacryl S-200 to fractionate water-insoluble protein by gel permeation chromatography was investigated. Elution of protein standards from colums of Sephacryl S-200 equilibrated with formamide, an excellent solvent for both hydrophobic and hydrophilic proteins, was molecular weight dependent. Advantages of chromatography of water-insoluble proteins using Sephacryl S-200 include rapidity, recovery of protein free of detergent or solvent, safety, ability to purify large amounts of protein, and ability to separate proteins as large as 100,000 daltons. Separation of water-insoluble polypeptides in a crude preparation of gliadin in formamide demonstrated the practicality of the method.

Hybridization of maize chloroplast DNA with transfer ribonucleic acids.

Hybridization of [125I] tRNA to chloroplast DNA indicates that 0.60-0.75% of maize chloroplast DNA contains sequences complementary to maize tRNA, corresponding to 20-26 tRNA cistrons. Green maize seedlings contain about twice the amount of chloroplast DNA-hybridizable tRNA as etiolated maize seedings. tRNA from green or etiolated maize seedlings was also aminoacylated in vitro with 21 labeled amino acids and then incubated with filters containing chloroplast DNA, tRNAs charging a total of at least 16 different amino acids hybridized with chloroplast DNA. Most of these plastid aminoacyl-tRNAs were present in higher concentrations in tRNA isolated from green maize seedlings, although there were several exceptions. The results are consistent with the hypothesis that a complete or nearly complete set or tRNAs can be transcribed from chloroplast DNA.

Poly(adenylic acid)-containing RNA from plastids of maize.

Hybridization experiments with chloroplast DNA and 125I-labeled RNA from maize seedlings suggest that chloroplasts and etioplasts contain detectable amounts of RNA that contains poly(adenylic aicd) (poly (A)) and was transcribed from chloroplast DNA. About 6% of the total poly(A)-containing RNA isolated from maize seedlings hybridized to chloroplast DNA. Poly(A)-containing RNA could also be isolated directly from purified chloroplasts that were treated with ribonucleases to reduce cytoplasmic contamination. At least 65% of this poly(A)-containing RNA hybridized to chloroplast DNA. Chloroplast poly(A) tracts average about 45 nucleotides in length, one-half the average size of poly(A) tracts from whole cells. The poly (A) tracts themselves are probably added to plastid RNAs following their transcription, because maize chloroplast DNA was found not to contain poly(dT).