Detection of mutations in genes by specific LNA primers

Details Detection of mutations in genes by specific LNA primers Detection of mutations in genes by specific LNA primers

2000-03-18

2001-11-13

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C536S023100, C536S024300

Reexamination Certificate

active

06316198

ABSTRACT:

FIELD OF THE INVENTION
The present invention comprises a method for detecting variant nucleic acids, a set of oligonucleotides suitable for this purpose, a reagent kit for performing the method of the invention and various uses and applications of the method in identifying specific gene sequences and diagnosing diseases and infections.
BACKGROUND OF THE INVENTION
Today, the testing of samples for the presence of certain nucleic acids in nucleic acid sequences gains increasingly more importance. This is partly due to the fact that the nucleotide sequence of a nucleic acid is a unique feature of each organism. First, a number of diseases are genetic in the sense that the nucleotide sequence for a “normal” gene is in some manner changed. Such a change could arise by the substitution of one base for another. Changes comprising more that one base can also be perceived. Given that three bases code for a single amino acid, a change in one base (a point mutation) could result in a change in the amino acid which, in turn, could result in a defective protein being made in a cell. Sickle cell anemia is a classic example of such a genetic defect caused by a change of a single base in a single gene; the beta-globin gene (A→T transversion at codon 6). Important point mutations can also be found for example in LDL receptors (Atherosclerosis (1992), 96:91-107) or in the apolipoprotein-B-gene (CGG→CAG mutation of codon 3500 (Proc. Natl. Acad. Sci. U.S.A. (1989) 86:587-591). Other examples of diseases caused by single gene defects include Factor IX and Factor VIII deficiency, breast cancer, cystic fibrosis, Factor V Leiden, Fragile X, Huntington disease, myotonic dystrophy, Haemophilia A, Haemophilia B, Neurofibromatosis type I, adenosine deaminase deficiency, purine nucleotide phosphorylase deficiency, ornithine transcarbamylase deficiency, argininsuccinate synthetase deficiency, beta-thalassemia, alpha-1 antitrypsin deficiency, glucocerebrosidase deficiency, phenylalanine hydroxylase deficiency and hypoxanthine-guanine phosphoribosyltransferase deficiency.
Second, the main cause of cancer is considered to be alterations in the cellular genes which directly or indirectly control cell growth and differentiation. There are at least thirty families of genes, called oncogenes, which are implicated in human tumor formation. Members of one such family, the ras gene family, are frequently found to be mutated in human tumors. In their normal state, proteins produced by the ras genes are thought to be involved in normal cell growth and maturation. Mutation of the ras gene, causing an amino acid alteration at one of three critical positions in the protein product, results in conversion to a form which is implicated in tumor formation. A gene having such a mutation is said to be “mutant” or “activated.” Unmutated ras is called “wild-type” or “normal” ras. It is thought that such a point mutation leading to ras activation can be induced by carcinogens or other environmental factors. Over 90% of pancreatic adenocarcinomas, about 50% of adenomas and adenocarcinomas of the colon, about 50% of adenocarcinomas of the lung and carcinomas of the thyroid, and a large fraction of haematological malignancies such as acute myeloid leukemia, lymphomas and myelodysplastic syndrome have been found to contain activated ras oncogenes. Overall, some 10 to 20% of human tumors have a mutation in one of the three ras genes (H-ras, Ki-ras, or N-ras).
Another example of a gene which is highly involved in the development of cancer is the TP53 gene. It is altered by mutations and/or deletions in more than half of all human cancers. The point mutations are scattered over more than 250 codons and mostly occur as missense mutations. In this respect, the TP53 gene differs from other tumor suppressor genes such as the retinoblastoma tumor suppressor gene (Rb1) and the p16 gene which are most frequently inactivated by deletions or nonsense mutations.
Most malignant tumors show alterations in both alleles of the TP53 gene. This usually involves the complete deletion of one allele and inactivation of the other allele by missense mutations. The result is either a complete lack of TP53 protein or expression of an altered protein. The missense mutations in the highly conserved regions of TP53 have also been associated with increased level of TP53 protein. This seems to result from mutation induced conformational changes, which stabilize the protein and extends its half-life from 4 to 8 hours. The majority of missense mutations cluster in the four highly conserved domains in the central core of the protein. This region is responsible for the sequence specific DNA binding and is therefore of critical importance for the functional integrity of TP53. Seven mutational hot spots have been identified within these domains. These are located at amino acid residues 175, 213, 245, 248, 249, 273, and 282.
Third, infectious diseases are caused by parasites, micro-organisms and viruses all of which have their own nucleic acids. The presence of these organisms in a sample of biological material is often determined by a number of traditional methods (e.g., culture). Each organism has its own unique genome and if there are genes or sequences of nucleic acids that are specific to a single species (to several related species, to a genus or to a higher level of relationship), this sequence will provide a “fingerprint” for that organism (or species, etc.), e.g. in the gene of the reverse transcriptase of the HIV virus (A→T mutation in codon 215: Science (1989) 246:1155-1158). Examples of other viruses include HPV, EBV, HSV, Hepatitis B and C and CMV. Examples of micro-organisms include bacteria and more particularly include various strains of mycoplasma, legionella, myco-bacteria, chlamydia, candida, gonocci, shigella and salmonella. As information on the genomes from more organisms are obtained by the scientific community the repertoire of micro-organisms that can be identified by the present invention will increase. In the nucleic acids of some bacterial strains, a particularly great similarity is found in the sequence of their ribosomal genes and their rRNA.
Current attempts in the field of examining samples for different nucleotide sequences focus on the use of only one single difference in the sequence of nucleotides in order to be able to discriminate between nucleic acids. Such differences may be a consequence of e.g. nucleotide exchanges caused by point mutation or in the case of micro-organisms a consequence of inter-species differences. Natural examples of such closely related nucleic acids are alleles, i.e. alternative variants of sequences of a given gene on a defined site on a chromosome.
In each example set forth above one can isolate nucleic acids from a sample and determine if the sample contains any of the above mentioned sequences, i.e. sequences specific for “genetic disease”, cancer, infectious diseases or infectious organisms, by identifying one or more sequences that are specific for a diseases or organism. A difficulty when identifying these differences or changes in the nucleotide sequence is however that the detection is not readily applicable in those instances where the number of copies of the target sequence present in a sample is low. In such instances it is difficult to distinguish signal from noise. One way around this problem is to increase the signal. Accordingly, a number of methods have been described to amplify the target sequences present in a sample. One of the best known and widely used amplification methods is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159.
Based upon the PCR technique a number of methods for detection of sequence variations have been described. From Oncogene Research 1 (1989), 235-241 and Nucl. Acids Res. 17 (1989), 8093-8099 a method is known where the area which presumably contains the allelic variant is first amplified in a PCR using specially designed primers and is then treated with a restriction e

Affiliated with

Also associated with

Rating

Detection of mutations in genes by specific LNA primers does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Detection of mutations in genes by specific LNA primers, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Detection of mutations in genes by specific LNA primers will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2599498