Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-03-11
2001-12-11
Zitomer, Stephanie W. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091200, C435S069100, C530S300000, C530S350000, C530S412000, C530S413000
Reexamination Certificate
active
06329180
ABSTRACT:
FIELD OF INVENTION
This invention relates to methods and kits for detecting mutations in genes by synthesizing in-vitro the proteins encoded by the genes with a peptide tag attached to the amino or carboxy termini, and analyzing the properties of the tagged proteins. In particular, this invention relates to the use of mass spectrometry for analyzing the tagged, and optionally purified proteins.
BACKGROUND OF THE INVENTION
Many genes that predispose to human disease have now been identified and hundreds more will be found over the next decade. In order to take advantage of these findings, the biomedical community must be able to identify individuals that carry mutations in these genes in order to counsel them and to help them take preventative measures.
The methods now being used to screen for mutations in certain disease causing genes suffer from short comings that will make it far too time consuming and expensive to carry out large scale screenings. For example, the breast cancer susceptibility genes BRCA1 and BRCA2 recently have been identified, and mutations predisposing a human to breast cancer have been found throughout the 16 kilobases of coding sequence contained in these two genes. The entire coding sequences of BRCA1 and BRCA2 are now routinely screened by DNA sequencing, Single Strand Conformation Polymorphism (SSCP), or the Protein Truncation Test (PTT). All three of these techniques rely on the electrophoretic properties of either DNA or protein, and detection of the electrophoresed products is accomplished by incorporation of a labeled tag. The label may be either a fluorescent or radioactive. Sequencing both strands of the 16 kb of BRCA½ for a single individual is costly and tedious with current techniques and gives a large amount of redundant information, since virtually all of the sequence will be identical to the known sequence. SSCP is less tedious, but still requires sequencing benign polymorphisms. Furthermore, SSCP segment length is limited to 150 base pairs, requiring a minimum of 6 PCR reactions per kilobase screened. PTT is the fastest of these techniques but it is incapable of detecting missense mutations, and also suffers from all of the drawbacks of gel electrophoresis, such as gel preparation, long running times, and low resolution.
Other techniques such as Allele Specific Oligomer Hybridization and Allele Specific PCR are designed to identify one previously characterized mutation per assay. These techniques are useful when a particular mutation has already been identified and is widespread, but cannot be used for genes where a large number of yet unidentified mutations exist in the human population.
Thus, a need exists for a reliable, high throughput method for gene mutation detection which takes advantage of the speed, sensitivity, accuracy and resolving power of analytical measurement instruments such as mass spectrometry instruments.
SUMMARY OF THE INVENTION
The present invention consists of a method for identifying genetic mutations by synthesizing proteins in-vitro and characterizing the protein product. The proteins are preferably synthesized with a peptide tag attached to their amino and/or carboxy terminus. In accordance with various embodiments of the method, the template for the in-vitro RNA and protein synthesis reactions can be a double stranded DNA polymerase chain reaction (PCR) product containing a test sequence of interest, although other forms of DNA can be used as well. When PCR products are used, the peptide tag can be encoded by a sequence present in either a 5′ primer or 3′ primer used to PCR amplify the test sequence. Any continuous stretch of coding sequence present in either genomic DNA or cDNA may be amplified with such primers, and the PCR product will have a sequence encoding the peptide tag in frame and adjacent to the test sequence. Any tagged test protein may be purified by means of an immobilized ligand specific for the tag, regardless of the composition of the test sequence.
The peptide tag may be used adjacent to any test sequence, and the assay can be multiplexed by incorporating different peptide tags with different test sequences so that different in-vitro synthesized proteins may be purified and analyzed separately.
Many ligands have been developed or discovered which bind with high affinity and specificity to particular peptides, among the most useful being monoclonal antibodies. Ligands of this type can be coupled to a solid support, for example, packed in an affinity chromatography column, and used to immunopurify peptide tagged proteins from complex mixtures rapidly and with high efficiency. Once the tagged test protein is purified from the other components of the cell lysate required for protein synthesis, it can be analyzed by various analytical instrument techniques, such as by mass spectrometry. In accordance with one embodiment, the purified protein is analyzed by the use of matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. Any single alteration in the DNA test sequence that alters the amino acid composition, and hence the mass of the protein product which it encodes, can be detected by the method of the present invention. One exception would be multiple alterations which cancel out each other's effects. However, such multiple mutations would be extremely rare. Also, leucine and isoleucine have the same mass, and an exchange of these residues will not alter the mass of the protein product.
The present invention provides several advantages. One important advantage is the elimination of the need to conduct electrophoretic separations of protein mixtures. In this manner, the speed and throughput of this method of analysis is significantly enhanced. Also, since the method provides a direct measure of mass, and not an inferred mass based upon electrophoretic mobility, mass accuracy is dramatically improved. Artifacts due to multiple protein folding states are completely eliminated, and mutations leading to amino acid substitutions are detectable by mass spectrometry.
These and other aspects, advantages, and objects of the present invention will be apparent from the following detailed description of the invention taken in conjunction with the drawings.
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Testa Hurwitz & Thibeault LLP
Tung Joyce
Zitomer Stephanie W.
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