Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-07
2003-05-06
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C436S094000
Reexamination Certificate
active
06558902
ABSTRACT:
FIELD OF THE INVENTION
The disclosed processes relate generally to the field of genomics, proteomics and molecular medicine, and more specifically to processes of using infrared matrix assisted laser desorption-ionization mass spectrometry to analyze, or otherwise detect the presence of or determine the identity of a biological macromolecule.
BACKGROUND OF THE INVENTION
In recent years, the molecular biology of a number of human genetic diseases has been elucidated by the application of recombinant DNA technology. More than 3000 diseases are known to be of genetic origin (Cooper and Krawczak, “Human Genome Mutations” (BIOS Publ. 1993)), including, for example, hemophilias, thalassemias, Duchenne muscular dystrophy, Huntington's disease, Alzheimer's disease and cystic fibrosis, as well as various cancers such as breast cancer. In addition to mutated genes that result in genetic disease, certain birth defects are the result of chromosomal abnormalities, including, for example, 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 Klinefelter's syndrome (XXY).
Other genetic diseases are caused by an abnormal number of trinucleotide repeats in a gene. These diseases include Huntington's disease, prostate cancer, spinal cerebellar ataxia 1 (SCA-1), Fragile X syndrome (Kremer et al.,
Science
252:1711-14 (1991); Fu et al.,
Cell
67:1047-58 (1991); Hirst et al.,
J. Med. Genet
. 28:824-29 (1991)); myotonic dystrophy type I (Mahadevan et al.,
Science
255:1253-55 (1992); Brook et al.,
Cell
68:799-808 (1992)), Kennedy's disease (also termed spinal and bulbar muscular atrophy (La Spada et al.,
Nature
352:77-79 (1991)), Machado-Joseph disease, and dentatorubral and pallidolyusian atrophy. The aberrant number of triplet repeats can be located in any region of a gene, including a coding region, a non-coding region of an exon, an intron, or a regulatory element such as a promoter. In certain of these diseases, for example, prostate cancer, the number of triplet repeats is positively correlated with prognosis of the disease.
Evidence indicates that amplification of a trinucleotide repeat is involved in the molecular pathology in each of the disorders listed above. Although some of these trinucleotide repeats appear to be in non-coding DNA, they clearly are involved with perturbations of genomic regions that ultimately affect gene expression. Perturbations of various dinucleotide and trinucleotide repeats resulting from somatic mutation in tumor cells also can affect gene expression or gene regulation.
Additional evidence indicates that certain DNA sequences predispose an individual to a number of other diseases, including diabetes, arteriosclerosis, obesity, various autoimmune diseases and cancers such as colorectal, breast, ovarian and lung cancer. Knowledge of the genetic lesion causing or contributing to a genetic disease allows one to predict whether a person has or is at risk of developing the disease or condition and also, at least in some cases, to determine the prognosis of the disease.
Numerous genes have polymorphic regions. Since individuals have any one of several allelic variants of a polymorphic region, each can be identified based on the type of allelic variants of polymorphic regions of genes. Such identification can be used, for example, for forensic purposes. In other situations, it is crucial to know the identity of allelic variants in an individual. For example, allelic differences in certain genes such as the major histocompatibility complex (MHC) genes are involved in graft rejection or graft versus host disease in bone marrow transplantation. Accordingly, it is highly desirable to develop rapid, sensitive, and accurate methods for determining the identity of allelic variants of polymorphic regions of genes or genetic lesions.
Several methods are used for identifying allelic variants or genetic lesions. For example, the identity of an allelic variant or the presence of a genetic lesion can be determined by comparing the mobility of an amplified nucleic acid fragment with a known standard by gel electrophoresis, or by hybridization with a probe that is complementary to the sequence to be identified. Identification only can be accomplished, however, if the nucleic acid fragment is labeled with a sensitive reporter function, for example, a radioactive (
32
P,
35
S), fluorescent or chemiluminescent reporter. Radioactive labels can be hazardous and the signals they produce can decay substantially over time. Non-radioactive labels such as fluorescent labels can suffer from a lack of sensitivity and fading of the signal when high intensity lasers are used. Additionally, labeling, electrophoresis and subsequent detection are laborious, time-consuming and error-prone procedures. Electrophoresis is particularly error-prone, since the size or the molecular weight of the nucleic acid cannot be correlated directly to its mobility in the gel matrix because sequence specific effects, secondary structures and interactions with the gel matrix cause artifacts in its migration through the gel.
Applications of mass spectrometry in the biosciences have been reported (see
Meth. Enzymol
., Vol. 193
, Mass Spectrometry
(McCloskey, ed.; Academic Press, NY 1990); McLaffery et al.,
Acc. Chem. Res
. 27:297-386 (1994); Chait and Kent,
Science
257:1885-1894 (1992); Siuzdak,
Proc. Natl. Acad. Sci., USA
91:11290-11297 (1994)), including methods for mass spectrometric analysis of biopolymers (see Hillenkamp et al. (1991)
Anal. Chem
. 63:1193A-1202A) and for producing and analyzing biopolymer ladders (see, International Publ. WO 96/36732; U.S. Pat. No. 5,792,664).
Mass spectrometry has been used for the analysis of nucleic acids (see, for example, Schram,
Mass Spectrometry of Nucleic Acid Components, Biomedical Applications of Mass Spectrometry
34:203-287 (1990); Crain,
Mass Spectrom. Rev
. 9:505-554 (1990); Murray,
J. Mass Spectrom. Rev
. 31:1203 (1996); Nordhoff et al.,
Mass Spectrom. Rev
. 15:67-138 (1997); U.S. Pat. No. 5,547,835; U.S. Pat. No. 5,605,798; PCT Application Publication No. W094/16101; PCT Application Publication No. WO 96/29431).
The so-called “soft ionization” mass spectrometric methods, including Matrix-Assisted Laser Desorption/Ionization (MALDI) and ElectroSpray Ionization (ESI), allow intact ionization, detection and mass determination of large molecules, i.e., well exceeding 300 kDa in mass (Fenn et al.,
Science
246:64-71 (1989); Karas and Hillenkamp,
Anal. Chem
. 60:2299-3001 (1988)). MALDI mass spectrometry (MALDI-MS; reviewed in Nordhoff et al.,
Mass Spectrom. Rev
. 15:67-138 (1997)) and ESI-MS have been used to analyze nucleic acids. Nucleic acids are very polar biomolecules that are difficult to volatilize and, therefore, there has been an upper mass limit for clear and accurate resolution.
ESI has been used for the intact desorption of large nucleic acids even in the megaDalton mass range (Ferstenau and Benner,
Rapid Commun. Mass Spectrom
. 9:1528-1538 (1995); Chen et al.,
Anal. Chem
. 67:1159-1163 (1995)). Mass assignment using ESI is very poor and only possible with an uncertainty of about 10%. The largest nucleic acids that have been accurately mass determined by ESI-MS are a 114 base pair double stranded PCR product (Muddiman et al.,
Anal. Chem
. 68:3705-3712 (1996)) of about 65 kDA in mass and a 120 nucleotide
E.coli
5S rRNA of about 39 kDa in mass (Limbach et al.,
J. Am. Soc. Mass Spectrom
. 6:27-39 (1995)). Furthermore, ESI requires extensive sample purification.
MALDI-MS requires incorporation of the macromolecule to be analyzed in a matrix, and has been performed on polypeptides and on nucleic acids mixed in a solid (i.e., crystalline) matrix. In these methods, a laser is used to strike the biopolymer/matrix mixture, which is crystallized on a probe tip, thereby effecting desorption and ionization of the biopolymer. In addition, MALDI-MS has been performed on polypeptides using the
Heller Ehrman White & McAuliffe LLP
Horlick Kenneth R.
Seidman Stephanie L.
Sequenom Inc.
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