Methods for multiplex detection of alleles associated with ophthalmic conditions
Systems and methods for detecting at least two genomic alleles associated with corneal dystrophy in a sample from a human subject are disclosed in which cells (e.g., epithelial) of the subject are adhered to a tip of a substrate. The tip of the substrate is agitated in a lysis solution that lyses cells adhered to the substrate. The substrate is removed from the lysis solution upon completion of this agitation. The resulting lysis solution is incubated and then genomic DNA from the lysis solution is isolated to form a gDNA solution. From this, identity of at least two nucleotides present in the human TGFβI gene is determined using at least two oligonucleotide primer pairs and the gDNA solution. These at least two nucleotides are located at respective independent positions of the TGFβI gene corresponding to respective independent single nucleotide polymorphisms (SNPs) associated with corneal dystrophy.
1. A reaction mixture for detecting corneal dystrophy in a human subject, the reaction mixture comprising:
a first labeled detection probe consisting of a first nucleic acid sequence consisting of SEQ ID NO: 25 with a label attached to the first nucleic acid sequence,
a second labeled detection probe consisting of a second nucleic acid sequence consisting of SEQ ID NO: 48 with a label attached to the second nucleic acid sequence,
a third labeled detection probe consisting of a third nucleic acid sequence consisting of SEQ ID NO: 49 with a label attached to the third nucleic acid sequence, and
a fourth labeled detection probe consisting of a fourth nucleic acid sequence consisting of SEQ ID NO: 42 with a label attached to the fourth nucleic acid sequence, wherein the only labeled detection probes in the reaction mixture are the first, the second, the third, and the fourth labeled detection probes.
2. The reaction mixture according to claim 1 , further comprising at least a first amplification primer pair for amplifying and determining a TGFβI gene sequence from a biological sample from the subject.
3. The reaction mixture according to claim 2 , wherein the first amplification primer pair comprises a first amplification primer and a second amplification primer, the first amplification primer comprises nucleotide sequence SEQ ID NO:1, and the second amplification primer comprises nucleotide sequence SEQ ID NO:2.
4. The reaction mixture according to claim 1 , wherein one or more of the first, second, third, and fourth labeled detection probes is coupled with a first label, one or more of the first, second, third, and fourth labeled detection probes is coupled with a second label, and the first and second labels are different.
5. The reaction mixture according to claim 4 , wherein the first label is VIC.
6. The reaction mixture according to claim 4 , wherein the second label is FAM.
7. The reaction mixture according to claim 4 , wherein one or more of the first, second, third, and fourth labeled detection probes further comprises a minor groove binder.
8. A method for detecting whether or not a human subject carries a mutation that causes corneal dystrophy comprising:
(a) amplifying a first TGFβ1 gene sequence from a biological sample from a human subject using a reaction mixture comprising at least a first amplification primer pair and the reaction mixture of claim 1 ;
(b) detecting whether or not a mutation is present in the first TGFβ1 gene sequence based on (i) the hybridization or (ii) lack of hybridization of the labeled detection probes in the reaction mixture.
9. The method of claim 8 , wherein the first amplification primer pair comprises SEQ ID NO: 1 and SEQ ID NO: 2.
10. The method of claim 8 , wherein one or more of the first, second, third, and fourth labeled detection probes is coupled with a first label, one or more of the first, second, third and fourth labeled probes is coupled with a second label, and the first and second labels are different.
11. The method of claim 10 , wherein the first label is VIC.
12. The method of claim 10 , wherein the second label is FAM.