Methods of preparing nucleic acids for mass spectrometric...

Details Methods of preparing nucleic acids for mass spectrometric... Methods of preparing nucleic acids for mass spectrometric...

1998-06-03

2003-05-20

Borin, Michael (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C250S281000

Reexamination Certificate

active

06566055

ABSTRACT:

INTRODUCTION
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 DNA sequence in 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 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. Wild type is a standard or reference nucleotide sequence to which variations are compared. As defined, any variation from wild type is considered a polymorphism including naturally occurring sequence variations and pathogenic mutations.
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 using the methods described here 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, which 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 typically the accuracy is only 5% to 10%. Also the molecular weight resolution is limited. In cases where two DNA fragments with identical number of base pairs can be separated, 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. 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.
All of the 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., “Detection of Polymorphisms of Human DNA by Gel Electrophoresis as Single-Stranded Conformation Polymorphisms,” Proc. Natl. Acad. Sci. USA 86, 2766 (1989)), denaturing gradient gel electrophoresis (DGGE) (Abrams et al., “Comprehensive Detection of Single Base Changes in Human Genomic DNA Using Denaturing Gradient Gel Electrophoresis and a GC Clamp,” Genomics 7, 463 (1990)), chemical cleavage at mismatch (CCM) (J. A. Saleeba & R. G. H. Cotton, “Chemical Cleavage of Mismatch to Detect Mutations,” Methods in Enzymology 217, 286 (1993)), enzymatic mismatch cleavage (EMC) (R. Youil et al., “Screening for Mutations by Enzyme Mismatch Cleavage with T4 Endonuclease VII,” Proc. Natl. Acad. Sci. USA 92, 87 (1995)), and “cleavase” fragment length polymorphism (CFLP) procedures are currently gel-based, making them cumbersome to automate and perform efficiently. There is a need for new methods that can provide cost effective and expeditious means for screening genetic material in an effort to reduce medical expenses. The inventions described here address these issues by developing novel, tailor-made processes that focus on the use of mass spectrometry as a genetic analysis tool. Mass spectrometry requires minute samples, provides extremely detailed information about the molecules being analyzed including high mass accuracy, and is easily automated.
The late 1980's saw the rise of two new mass spectrometric techniques for successfully measuring the masses of intact very large biomolecules, namely, matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) (K. Tanaka et al., “Protein and Polymer Analyses up to m/z 100,000 by Laser Ionization Time-of-flight Mass Spectrometry,” Rapid Commun. Mass Spectrom. 2, 151-153 (1988); B. Spengler et al., “Laser Mass Analysis in Biology,” Ber. Bunsenges. Phys. Chem. 93, 396-402 (1989)) and electrospray ionization (ESI) combined with a variety of mass analyzers (J. B. Fenn et al., Science 246, 64-71 (1989)). Both of these two methods are suitable for genetic screening tests. 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. One of the advances in MALDI analysis of polynucleotides was the discovery of 3-hydroxypicolinic acid as a matrix for mixed-base oligonucleotides. Wu, et al., Rapid Comm'ns in Mass Spectrometry, 7:142-146 (1993).
MALDI-TOF MS involves laser pulses focused on a small sample plate comprising analyte molecules (nucleic acids) embedded in either a solid or liquid matrix comprising a small, highly absorbing compound. 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. If double-stranded nucleic acids are analyzed, the MALDI-TOF MS typically results in mostly denatured single-strand detection. The ions generated by the laser pulses are accelerated to a fixed kinetic energy by a strong electric field and then pass through an electric field-free region in vacuum in which the ions travel with a velocity corresponding to their respective mass-to-charge ratios (m/z). The smaller m/z ions will travel through the vacuum region faster than the larger m/z ions thereby causing a separation. At the end of the electric field-free region, the ions collide with a detector that generates a signal as each set of ions of a particular mass-to-charge ratio strikes the detector. Usually for a given assay, 10 to 100 mass spectra resulting from individual laser pulses are summed together to make a single composite mass spectrum with an improved signal-to-noise ratio.
The mass of an ion (such as a charged nucleic acid) is measured by using its velocity to determine the mass-to-charge ratio by time-of-flight analysis. In other words, the mass of the mol

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