Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2000-02-29
2001-07-24
Nguyen, Kiet T. (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
Reexamination Certificate
active
06265716
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to volatile photoabsorbing matrices having a low sublimation temperature for use in the mass spectrometric analysis of large, nonvolatile molecules. This invention also relates to methods for preparing samples containing large, nonvolatile analyte molecules for laser desorption mass spectrometry employing such matrices.
2. Description of Related Art
Approximately 4,000 human disorders are attributed to genetic causes. Hundreds of genes responsible for various disorders have been mapped, and sequence information is being accumulated rapidly. A principal goal of the Human Genome Project is to find all genes associated with each disorder. The definitive diagnostic test for any specific genetic disease (or predisposition to disease) will be the identification of polymorphic variations in the DNA sequence of affected cells that result in alterations of gene function. Furthermore, response to specific medications may depend on the presence of polymorphisms. Developing DNA (or RNA) screening as a practical tool for medical diagnostics requires a method that is inexpensive, accurate, expeditious, and robust.
Genetic polymorphisms and mutations can manifest themselves in several forms, such as point polymorphisms or point mutations where a single base is changed to one of the three other bases; deletions where one or more bases are removed from a nucleic acid sequence and the bases flanking the deleted sequence are directly linked to each other; insertions where new bases are inserted at a particular point in a nucleic acid sequence adding additional length to the overall sequence; and expansions and reductions of repeating sequence motifs. Large insertions and deletions, often the result of chromosomal recombination and rearrangement events, can lead to partial or complete loss of the activity of a gene. Of these forms of polymorphism, in general the most difficult type of change to screen for and detect is the point polymorphism because it represents the smallest degree of molecular change.
Although a number of genetic defects can be linked to a specific single point mutation within a gene, e.g. sickle cell anemia, many are caused by a wide spectrum of different mutations throughout the gene. A typical gene that might be screened could be anywhere from 1,000 to 100,000 bases in length, though smaller and larger genes do exist. Of that amount of DNA, only a fraction of the base pairs actually encode the protein. These discontinuous protein coding regions are called exons and the remainder of the gene is referred to as introns. Of these two types of regions, exons often contain the most important sequences to be screened. Several complex procedures have been developed for scanning genes in order to detect polymorphisms. These procedures are applicable to both exons and introns.
In terms of current use, most of the methods to scan or screen genes employ slab or capillary gel electrophoresis for the separation and detection step in the assays. Gel electrophoresis of nucleic acids primarily provides relative size information based on mobility through the gel matrix. If calibration standards are employed, gel electrophoresis can be used to measure absolute and relative molecular weights of large biomolecules with some moderate degree of accuracy; even then, the accuracy is typically only 5% to 10%. Also the molecular weight resolution is limited. In cases where two DNA fragments with the identical number of base pairs can be separated, for example, by using high concentration polyacrylamide gels, it is still not possible to identify which band on a gel corresponds to which DNA fragment without performing secondary labeling experiments. Thus, gel electrophoresis techniques can only determine size and cannot provide any information about changes in base composition or sequence without performing more complex sequencing reactions. Gel-based techniques, for the most part, are dependent on labeling or staining methods to visualize and discriminate between different nucleic acid fragments.
Many methods in use today capable of screening broadly for genetic polymorphisms suffer from technical complication and are labor and time intensive. Single strand conformational polymorphism (SSCP) (Orita et al., 1989), denaturing gradient gel electrophoresis (DGGE) (Abrams et al., 1990), chemical cleavage at mismatch (CCM) (Saleeba and Cotton, 1993), enzymatic mismatch cleavage (EMC) (Youil et al., 1995), and cleavage fragment length polymorphism (CFLP) procedures are currently gel-based, making them cumbersome to automate and perform efficiently. Thus, there is a need for new methods that can provide cost effective and expeditious means for screening genetic material in an effort to detect genetic mutations and diagnose related medical conditions simply, quickly, accurately, and inexpensively.
Another approach that is having some success is to employ mass spectrometry to screen for and detect genetic mutations as well as to sequence nucleic acids. In order to measure the mass of nonvolatile high molecular weight molecules, typically greater than 1000 Da, in a mass spectrometer, the analyte molecules must first be volatilized or converted into gas-phase ions. Although direct laser desorption of the neat analyte is one approach to volatilizing the molecule, the energy deposited into the analyte may induce fragmentation and lead to results that are ambiguous or difficult to analyze. The late 1980's saw the rise of two new mass spectrometric techniques which are potentially suitable for genetic screening tests by successfully measuring the masses of intact very large biomolecules, namely, matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) (Tanaka et al., 1988; Spengler et al., 1989) and electrospray ionization (ES) combined with a variety of mass analyzers. The MALDI mass spectrometric technique can also be used with methods other than time-of-flight, for example, magnetic sector, Fourier-transform ion cyclotron resonance, quadrupole, and quadrupole trap.
MALDI-TOF MS involves laser pulses focused on a small sample plate on which analyte molecules (i.e. nucleic acids) are embedded in either a solid or liquid matrix which is typically a small, highly absorbing material, such as a small aromatic organic molecule. The volatilization of intact fragile molecules benefits from the use of matrix-assisted laser desorption ionization because the radiative energy from the laser pulse is coupled indirectly into the analyte through the matrix molecules. Typically, the analyte molecules are crystallized with a large molar excess of a photoabsorbing matrix (see U.S. Pat. Nos. 4,920,264 and 5,118,937, incorporated herein by reference). An advance in MALDI analysis of polynucleotides was the discovery of 3-hydroxypicolinic acid (3-HPA) as a suitable matrix for mixed-base oligonucleotides (Wu, et al., 1993).
The laser pulses transfer energy to the matrix causing a microscopic ablation and concomitant ionization of the analyte molecules, producing a gaseous plume of intact, charged nucleic acids in single-stranded form. It is thought that upon laser excitation the matrix molecules are rapidly heated and ejected into the gas phase, carrying analyte molecules into the expansion plume of molecules and ions. It is thought that gas-phase ion-molecule collisions subsequently ionize the neutral analyte molecules in the near-surface region, often via proton transfer. The matrix thus functions as both an energy- and charge-transfer species. If double-stranded nucleic acids are analyzed, the MALDI-TOF MS typically results in detection of mostly charged denatured single-stranded nucleic acids.
The ions generated by the laser pulses are accelerated to a fixed kinetic energy by a strong electric field and then passed through an electric field-free region in vacuum, traveling with a velocity corresponding to their respective mass-to-charge ratios (m/z). Thus, the smaller m/z ions will travel through the vacuum region f
Becker Christopher H.
Hunter Joanna M.
Lin Hua
Genetrace Systems, Inc.
Horne Angela P.
Law Offices of Jonathan Alan Quine
Nguyen Kiet T.
Quine Jonathan Alan
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