Methods and apparatus for screening and detecting multiple genetic mutations
View Patent ↗An assay system and methods are described where patient samples containing genomic DNA are analyzed for the presence of known genetic polymorphisms using a universal reporter strategy. In a preferred embodiment, the amplified DNA is localized at test sites in an array of sites on a microchip followed by a series of hybridization reactions that screen for the presence of a single mutation from among a number of mutations, and allow the identification of specific mutations. In addition to universal reporters, the assay may use blockers and discriminators for screening and identification of known polymorphisms.
1. A method for detecting a polymorphism related to a genetic disease in a patient sample nucleic acid, comprising the steps of:
providing the patient sample nucleic acid containing a first locus having a first polymorphism and a second locus having a second polymorphism on a site of a microarray, wherein the first or second polymorphism is related to the genetic disease;
providing an unlabeled blocker that is complementary to the first locus containing the first polymorphism;
hybridizing the unlabeled blocker with the first locus such that the first polymorphism is blocked by the unlabeled blocker and the second locus is unblocked;
providing a detectable discriminator that is capable of hybridizing with the second locus containing the second polymorphism and specifically identifying the second polymorphism;
hybridizing the detectable discriminator with the second locus containing the second polymorphism; and
detecting the second polymorphism by detecting the presence of the discriminator at the site of the microarray.
2. The method of claim 1 , wherein the site of the microarray comprises a site of an actively addressable electronic microarray.
3. The method of claim 2 , wherein the addressable electronic microarray includes a permeation layer.
4. The method of claim 2 , wherein the microarray further comprises multiple sites and multiple patient nucleic acid samples are provided on the multiple sites of the microarray.
5. The method of claim 1 , wherein the patient sample nucleic acid is amplified.
6. The method of claim 5 , wherein the amplification includes polymerase chain reaction (PCR).
7. The method of claim 5 , wherein the amplification includes ligase chain reaction (LCR).
8. The method of claim 5 , wherein the amplification includes strand displacement amplification (SDA).
9. The method of claim 5 , wherein the amplification is performed using transcription-based amplification system (TAS).
10. The method of claim 5 , wherein the amplification is performed using self-sustained sequence replication system (3 SR).
11. The method of claim 5 , wherein the amplification is performed using Qβ replicase amplification system (Qβ).
12. The method of claim 1 , further includes the step of performing a screening step for a number of patient nucleic acid samples.
13. The method of claim 1 , wherein the patient sample nucleic acid comprises multiple segments containing different loci.
14. The method of claim 13 , wherein the multiple segments containing different loci are affixed to an identical site of the microarray.
15. The method of claim 13 , wherein the multiple segments containing different loci are affixed to different sites of the microarray.
16. The method of claim 1 , further comprising the steps of:
providing a labeled amplification control that is capable of binding with the patient nucleic acid sample; and
hybridizing the labeled amplification control to the patient nucleic acid sample.
17. The method of claim 1 , wherein the genetic disease is cystic fibrosis.