Methods for the detection of nucleic acid differences
The invention provides methods permitting the detection of small amounts of different nucleic acids in the presence of an excess amount of wild-type nucleic acids. Also provided herein are method so detecting infectious disease minority variants, methods of forensic identification, methods of diagnosing cancer and monitoring disease progress.
1. A method for detecting the presence of pancreatic cancer or chronic pancreatitis in a human subject, the method comprising:
obtaining a sample of pancreatic juice from the subject, the sample comprising nucleic acids from the subject;
creating a reaction mixture by hybridizing the nucleic acids to a plurality of oligonucleotide pairs to detect both wild type KRAS2 and mutant KRAS2 sequences in the nucleic acid to form a reaction mixture, wherein each oligonucleotide pair:
comprises a first oligonucleotide and a second oligonucleotide, wherein each first oligonucleotide comprises a first gene specific region and a first primer region, each second oligonucleotide comprises a second gene specific region and a second primer region, and either the first oligonucleotide or the second oligonucleotide comprises a detection region; and in oligonucleotide pairs to detect mutant KRAS2 sequence either the first oligonucleotide or the second oligonucleotide comprises a nucleotide in the gene specific region that detects one of a G35A, a G35T, or a G34C nucleotide substitution in the KRAS2 sequence; and in each oligonucleotide pair the first oligonucleotide and the second oligonucleotide are suitable for ligation to one another;
subjecting the reaction mixture to a ligation reaction to form ligation products from said oligonucleotide pairs to detect both wild type KRAS2 and mutant KRAS2 sequences;
amplifying said ligation products to form wild type KRAS2 and mutant KRAS2 amplification products;
detecting the quantity of the wild type KRAS2 and mutant KRAS2 amplification products;
calculating a mutation level, wherein said mutation level is equal to quantity of mutant KRAS2/(quantity of mutant KRAS2+quantity of wild-type KRAS2); and
detecting a mutation level that is less than 0.5% and correlating the mutation level that is less than 0.5% with the presence of chronic pancreatitis in the subject; or detecting a mutation level that is greater than 0.5% and less than 80% and correlating the mutation level that is greater than 0.5% and less than 80% with the presence of pancreatic cancer in the subject.
2. The method of claim 1 , wherein a mutation level that is less than 0.5% is detected.
3. The method of claim 1 , wherein a mutation level that is greater than 0.5% and less than 80% is detected.
4. The method of claim 1 , further comprising monitoring KRAS mutation levels.
5. The method of claim 1 , wherein the sensitivity is at least 76%.
6. The method of claim 1 , wherein the sensitivity is at least 88%.
7. The method of claim 1 , wherein the sensitivity is 94%.
8. The method of claim 1 , wherein a first oligonucleotide or a second oligonucleotide comprises a foreign DNA region between the gene specific region and the primer region.
9. The method of claim 8 , wherein the foreign DNA region is used as a probe.
10. The method of claim 1 , wherein amplification comprises a method selected from the group consisting of the amplifying is by quantitative polymerase chain reaction (PCR), real time PCR, and real time quantitative PCR.
11. The method of claim 10 , wherein the amplifying comprises a multiplex PCR reaction.