Detection of allele - specific mutagens
AssigneeTrustees of Darmouth College
InventorGeorge D. Sorenson
Life of this patent
Estimating the term…
Abstract
Methods are provided for detecting and quantitating gene sequences, such as mutated genes and oncogenes, in biological fluids. The fluid sample (e.g., plasma, serum, urine, etc.) is obtained, deproteinized and the DNA present in the sample is extracted. Following denaturation of the DNA, an amplification procedure, such as PCR or LCR, is conducted to amplify the mutated gene sequence.
Claims21
A method of detecting a mutant allele, comprising the steps of:
- providing a sample of a biological fluid containing soluble DNA, including a mutant allele of interest;
- deproteinizing the DNA and substantially simultaneously therewith inactivating any DNases present in the sample;
- extracting the DNA from the sample;
- denaturing the DNA to dissociate first and second strands of the DNA;
- amplifying the mutant allele of interest in an allele-specific manner using at least a first set of four allele-specific oligodeoxynucleotide primers having one primer the 3' terminal nucleotide of which is complementary to a mutation-containing segment on a first strand of the DNA and a first common oligodeoxynucleotide primer for pairing during amplification to each allele-specific oligodeoxynucleotide primer, the common oligodeoxynucleotide primer being complementary to a segment of a second strand of the DNA distant with respect to the position of the first oligodeoxynucleotide primer;
- and
- detecting the presence of the mutant allele of interest.
A diagnostic kit for detecting the presence of a K-ras mutation in the nucleic acids in biological fluid, wherein the mutation is present in the twelfth codon at position 1, comprising:
- reagents to facilitate the deproteinization and isolation of DNA;
- reagents to facilitate amplification by the polymerase chain reaction;
- a heat stable DNA polymerase;
- and
- a first set of allele-specific oligodeoxynucleotide sense primers having the following sequences
- SEQ ID NO:
- 1
- SEQ ID NO:
- 2
- SEQ ID NO:
- 3
- SEQ ID NO 4
- and a first common oligodeoxynucleotide antisense primer having the following sequence
- SEQ ID NO:
- 5.
Dependencies are inferred from the claim text.