Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-18
2002-08-20
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200
Reexamination Certificate
active
06436640
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to DNA diagnostics based on mass spectrometry where fully or partially LNA modified DNA probes are used in order to enhance stability and resolution.
BACKGROUND OF THE INVENTION
The genetic information of all living organisms (e.g. animals, plants and micro-organisms) is encoded in deoxyribonucleic acid (DNA). In humans, the complete genome is comprised of about 100,000 genes located on 24 chromosomes (The Human Genome, T. Strachan, BIOS Scientific Publishers, 1992). Each gene codes for a specific protein which after its expression via transcription and translation, fulfils a specific biochemical function within a living cell. Changes in a DNA sequence are known as mutations and can result in proteins with altered or in some cases even lost biochemical activities; this in turn can cause genetic disease. Mutations include nucleotide deletions, insertions or alterations (i.e. point mutations). Point mutations can be either “mis-sense”, resulting in a change in the amino acid sequence of a protein or “nonsense” coding for a stop codon and thereby leading to a truncated protein. Furthermore, the detection of polymorphisms might be equally interesting.
More than 3000 genetic diseases are currently known (Human Genome Mutations, D. N. Cooper and M. Krawczak, BIOS Publishers, 1993), including hemophilias, thalassemias, Duchenne Muscular Dystrophy (DMD), Huntington's Disease (HD), Alzheimer's Disease and Cystic Fibrosis (CF). In addition to mutated genes, which result in genetic disease, certain birth defects are the result of chromosomal abnormalities such as Trisomy 21 (Down's Syndrome). Trisomy 13 (Patau Syndrome), Trisomy 18 (Edward's Syndrome), Monosomy X (Turner's Syndrome) and other sex chromosome aneuploidies such as Klienfelter's Syndrome (XXY). Further, there is growing evidence that certain DNA sequences may predispose an individual to any of a number of diseases such as diabetes, arteriosclerosis, obesity, various autoimmune diseases and cancer (e.g. colorectal, breast, ovarian, lung). Viruses, bacteria, fungi and other infectious organisms contain distinct nucleic acid sequences, which are different from the sequences contained in the host cell. Therefore, infectious organisms can also be detected and identified based on their specific DNA sequences.
Since the sequence of about 16 nucleotides is specific on statistical grounds even for the size of the human genome, relatively short nucleic acid sequences can be used to detect normal and defective genes in higher organisms and to detect infectious microorganisms (e.g. bacteria, fungi, protists and yeast) and viruses. DNA sequences can serve as a fingerprint for detection of different individuals within the same species.
Analysis of nucleic acid molecules by mass spectrometry has thus met increasing interest in recent years.
In general, mass spectrometry provides a means of “weighing” individual molecules by ionising the molecules in vacuo and making them “fly” by volatilisation. Under the influence of combinations of electric and magnetic fields, the ions follow trajectories depending on their individual mass (m) and charge (z). In the range of molecules with low molecular weight, mass spectrometry has long been part of the routine physical-organic repertoire for analysis and characterisation of organic molecules by the determination of the mass of the parent molecular ion. In addition, by arranging collisions of this parent molecular ion with other particles (e.g. argon atoms), the molecular ion is fragmented forming secondary ions by the so-called collision induced dissociation (CID). The fragmentation pattern/pathway very often allows the derivation of detailed structural information. Many applications of mass spectrometric methods are known in the art, particularly in biosciences, and can be found summarised in
Methods in Enzymology
, Vol. 193:“Mass Spectrometrv” (J. A. McCloskey, editor), 1990, Academic Press, New York and “Mass spectrometry for Biotechnology” by G. Siuzdak, 1996, Academic Press.
Two more recent ionisation/desorption techniques are electrospray/ionspray (ES) and matrix-assisted laser desorption/ionisation (MALDI). ES mass spectrometry has been introduced by Fenn et al. (
J. Phys. Chem
. 88, 4451-59 (1984); PCT Application No. WO 90/14148) and current applications are summarised in recent review articles (R. D. Smith et al., Anal. Chem., 62, .882-89 (1990) and B. Ardrey. Electrospray Mass Spectrometry,
Spectroscopy Europe
, 4, 10-18 (1992)). As a mass analyser, a quadrupole is most frequently used. The determination of molecular weights in femtomole amounts of sample is very accurate due to the presence of multiple ion peaks which all could be used for the mass calculation.
MALDI mass spectrometry, in contrast, can be particularly attractive when a time-of-flight (TOF) configuration is used as a mass analyser. The MALDI-TOF mass spectrometry has been introduced by Hillenkamp et al. (“Matrix Assisted UV-Laser Desorption/Ionisation: A New Approach to Mass Spectrometry of Large Biomolecules.”
Biological Mass Spectrometry
(Burlingame and McCloskey, editors), Elsevier Science Publishers, Amsterdam, pp. 49-60, 1990). Since, in most cases, no multiple molecular ion peaks are produced with this technique, the mass spectra, in principle, look simpler compared to ES mass spectrometry.
WO 94/16101 (Koster) describes DNA sequencing by means of mass spectrometry and WO 94/21822 (Koster) describes DNA sequencing by means of mass spectrometry via exonuclease degradation.
WO 96129431 (Sequenom) describes various ways of determining the sequence of and mutations in nucleic acid molecules by means of mass spectrometry analysis. It is described that introduction of mass differentiating markers facilitates the resolution of the mass spectrometry signals and thereby allows for multiplexing.
Bicyclic nucleotide analogues, i.e. nucleotide analogues containing bicyclic sugars, have been described in the literature. Substitution of natural nucleotides in oligonucleotides with bicyclic analogues has, however, often been performed at the cost of specificity or affinity.
Recently, however, Locked Nucleoside Analogues (LNA) have been described by Wengel and co-workers and others (see e.g. Nielsen et al., J. Chem. Soc. Perkin Trans. 1, 1997, p 3423; Koshkin et al., Tetrahedron 54 (1998), p 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett. 8 (1998) 2219-2222; and Obika et al., Tetrahedron Lett. 39 (1998), 5401-5404), and it has been described that novel bicyclic nucleotide analogues exhibit improved affinity and specificity characteristics when incorporated into oligonucleotides. It is believed that a whole class of such modified nucleotide analogues possess the characteristics described by Wengel and co-workers.
SUMMARY OF THE INVENTION
The present applicants have found that the use of LNA modified oligonucleotides in processes for the detection of a target nucleic acid sequence of a nucleic acid molecule or for the detection of a mutation in a nucleic acid sequence of a nucleic acid molecule by mass spectrometry offers at least three major advantages, namely (a) the possibility of adjusting (typically increasing) the specificity and/or affinity of an oligonucleotide involved in the detection process for the nucleic acid molecule, (b) the direct inclusion of mass differentiation markers, and (c) increased stability of the LNA modified oligonucleotides under conditions of mass spectrometry.
Thus, the present invention provides a process for detecting a target nucleic acid sequence of a nucleic acid molecule or for detecting a mutation in a nucleic acid sequence of a nucleic acid molecule, wherein (a) the nucleic acid molecule or (b) a part of the nucleic acid molecule or (c) an oligonucleotide complementary to the sequence or at least a sub-sequence of the nucleic acid molecule is analysed by mass spectrometry in order to obtain direct or indirect information about said target nucleic acid sequence or mutation, and wherein the process involves th
Simmons Adrian
Smith Clifford
Corless Peter F.
Edwards & Angell LLP
Exiqon A/S
Horlick Kenneth R.
Rees Dianne M.
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