US20230348984A1

US20230348984A1 - Methods for multiplex detection of alleles associated with ophthalmic conditions

Info

Publication number: US20230348984A1
Application number: US18/064,444
Authority: US
Inventors: Connie CHAO-SHERN; Sun-Young CHO; Gene Lee
Original assignee: Avellino Lab USA Inc
Current assignee: Avellino Lab USA Inc
Priority date: 2013-11-15
Filing date: 2022-12-12
Publication date: 2023-11-02
Also published as: TW201610167A; KR102293785B1; CN105899681B; JP6672147B2; KR20210099204A; US11525160B2; WO2015073978A2; JP7077289B2; TW202346604A; EP3872192A1; KR20160079117A; TW202223098A; AU2014348279A9; KR102483783B1; TWI812047B; CN105899681A; EP3068908B1; AU2014348279B2; US20160319357A1; SG10201804076RA

Abstract

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.

Description

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No. 15/154,895, filed May 13, 2016 (now U.S. Pat. No. 11,525,160), which is a continuation application of International Application No. PCT/US2014/065975, filed Nov. 17, 2014, which claims priority to U.S. Provisional Patent Application Ser. No. 61/905,051, filed Nov. 15, 2013. All of these patent applications are incorporated by reference herein in their entireties.

SEQUENCE LISTING SUBMISSION VIA EFS-WEB

The contents of the electronic sequence listing (070335-5007-US01_Sequence_Listing.xml; Size: 101,518 bytes; and Date of Creation: Jul. 20, 2023) is herein incorporated by reference in its entirety.

FIELD OF THE APPLICATION

This application generally relates to methods for the isolation and detection of disease-associated genetic alleles. In particular, this application relates to an improved method for the detection of an Avellino corneal dystrophy associated allele.

BACKGROUND

Real-time PCR can be used to detect differences between nucleic acid sequences having substantially identical sequences. Through the use of differentially labeled fluorescent nucleic acid probes, for example one that binds to a wild type sequence and one that binds to a mutant sequence, single nucleotide changes in the human genome can be quickly and reliably detected. This resolving power has been applied to medical diagnostics, where single nucleotide polymorphisms (SNPs), i.e., single base changes found within the coding and/or non-coding sequence of a protein, are correlated to human disease.
However, real-time PCR analysis is highly dependent upon the collection and isolation of high quality samples. Poor sample collection and/or isolation require the use of longer assay conditions and greater amounts of real-time PCR reagents, both of which result in increased costs and reduced productivity. Furthermore, failure of a real-time PCR single nucleotide polymorphism detection assay can result in the need to collect additional samples, causing even greater loss in time and resources.
Accordingly, methods resulting in improved sample collection and isolation, which improve the overall success rate of the assay, reduce the reagents required for the assay, and reduce the need to collect additional samples at later time are highly desirable. Furthermore, methods for performing real-time PCR SNP detection assays with lower amounts of sample material will also reduce the challenges associated with the collection and isolation of high quality samples.
Corneal dystrophy can be an autosomal dominant hereditary disease, which initially presents with blurred vision in the center of a patient's cornea. The blurry vision gradually spreads toward the perimeter of cornea, worsening the patient's vision as they age. There are several types of corneal dystrophy that have been characterized, including Avellino corneal dystrophy, Granular corneal dystrophy, lattice type I corneal dystrophy, Thiel-Behnke, and Reis-bucklers corneal dystrophy. Corneal dystrophies are known to be caused, at least in some cases, by mutations in the transforming growth factor beta induced (TGFβI) gene encoding the βIG-H3 protein (also known as TGFβI protein, TGFBI protein, and keratoepithelin).
Heterozygous patients suffering from Avellino corneal dystrophy have increasing loss in vision with age, becoming severe in the later years of life. Homozygous patients, in contrast, present with severe to complete loss of vision by six years of age. Avellino corneal dystrophy was first recognized as a distinct type of corneal dystrophy around 1988. Prior to then, it was likely misclassified as Granular corneal dystrophy. Today, Avellino corneal dystrophy is known to be the most common form of stromal corneal dystrophy world-wide. In Korea, Avellino corneal dystrophy is believed to have a prevalence around 1 in 870 people (see Lee, J. H. et al., Ophthalmic Epidemiol., 17:160, 2010; see also Holland, E. J. et al., Ophthalmology, 99:1564, 1992; Kennedy, S. M. et al., Br. J. Ophthalmol., 80:489, 1996; Dolmetsch, A. M. et al., Can. J. Ophthalmol., 31:29, 1996; Afshari, N. A. et al., Arch. Ophthalmol., 119:16, 2001; Stewart, H. S. Hum. Mutat., 14:126, 1999).
Previously, it was discovered that heterozygous individuals (e.g., having one wild type βIG-H3 allele and one mutant βIG-H3 allele) were highly susceptible to accelerating loss of vision following LASIK surgery. Notably, two years after surgery increased opacity of the cornea was observed in these patients with increasing aggressiveness, eventually resulting in complete loss of vision (Jun, R. M. et al., Opthalmology, 111:463, 2004). Previously, eye surgery has been performed with an expectation that LASIK or Excimer Laser surgery would get rid of vision blurriness of a patient suffering from corneal dystrophy. For a hypothetical number of three hundred thousand cases of LASIK surgery, 300 people would have lost their vision, based on 1/1000 of minimum estimation of heterozygous patients suffering from Avellino corneal dystrophy. Patients who have undergone LASIK surgery are mainly in their 20's and 30's carrying out productive activities; therefore, their vision loss causes serious troubles in both society and economics.
In addition, after approval of LASIK surgery in year 2000 in USA, African American patients suffering from Avellino corneal dystrophy who underwent LASIK surgery have been found to lose eye sight, which infers that plenty of similar cases might be occurring throughout the world.
Therefore, although accurate diagnosis of Avellino corneal dystrophy is required to prevent the progression of Avellino corneal dystrophy by LASIK surgery, the diagnosis of Avellino corneal dystrophy is just conducted by microscopic observation (e.g., slit-lamp examination) of corneal opacity and thus often doctors miss latent symptoms of patients to perform LASIK surgery, which results in vision loss. Therefore, rapid and precise genetic diagnosis of corneal dystrophy is desirable.
A DNA chip for detecting a mutation in βIG-H3 gene, which is responsible for Avellino corneal dystrophy, was developed (Korean Patent Laid-Open Publication No. 10-2007-0076532). However, the diagnosis of Avellino corneal dystrophy using the DNA chip disadvantageously requires several steps, including a step of amplifying DNA in a sample, a step of hybridizing the amplified DNA with the DNA chip, a step of washing the hybridized DNA chip, and a step of detecting a positive response.
Given the above background, what is needed in the art are improved methods for detecting multiple mutated alleles associated with corneal dystrophy.

SUMMARY

The present disclosure provides improved methods for the detection of one or more alleles associated with human disease. The methods described below decrease the time and cost associated with performing assays that yield medical information about a subject. For example, in some embodiments, the improved methods allow for same-day detection of a genomic marker associated with Avellino corneal dystrophy, at a reduced cost to the patient.
In some embodiments, the present disclosure provides methods for detecting at least two genomic alleles associated with corneal dystrophy in a sample from a subject, the method comprising: (A) providing epithelial cells of a subject adhered to a tip of a substrate; (B) agitating the tip of the substrate in a lysis solution that lyses cells adhered to the substrate; (C) removing the substrate from the lysis solution upon completion of the agitating (B); (D) incubating the lysis solution after the removing (C); (E) isolating genomic DNA from the lysis solution to form a gDNA solution; and (F) determining an identity of at least two nucleotides present in the TGFβI gene using at least two oligonucleotide primer pairs and the gDNA solution, wherein the 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.
In some embodiments, the at least two nucleotides present in the TGFβI gene are separated in the human genome by at least one nucleotide.
In some embodiments, at least one pair of the at least two oligonucleotide primer pairs comprises a forward PCR primer having a nucleotide sequence comprising SEQ ID NO:1 and a reverse PCR primer having a nucleotide sequence comprising SEQ ID NO:2.
In some embodiments, at least one pair of the at least two oligonucleotide primer pairs comprises a forward PCR primer having a nucleotide sequence comprising SEQ ID NO:43 and a reverse PCR primer having a nucleotide sequence comprising SEQ ID NO:44.
In some embodiments, the at least two oligonucleotide primer pairs comprise a first amplification primer pair and a second amplification primer pair. The first amplification primer pair comprises a forward PCR primer having a nucleotide sequence comprising SEQ ID NO:1 and a reverse PCR primer having a nucleotide sequence comprising SEQ ID NO:2. The second amplification primer pair comprises a forward PCR primer having a nucleotide sequence comprising SEQ ID NO:43 and a reverse PCR primer having a nucleotide sequence comprising SEQ ID NO:44.
In some embodiments, the determining (F) further comprises using: (i) a first wild type detection probe having a nucleotide sequence comprising SEQ ID NO:25 and a first mutant detection probe having a nucleotide sequence comprising SEQ ID NO:26, SEQ ID NO:48, or SEQ ID NO:49; and (ii) a second wild type detection probe having a nucleotide sequence comprising SEQ ID NO:45 or SEQ ID NO:47 and a second mutant detection probe having a nucleotide sequence comprising SEQ ID NO:46 or SEQ ID NO:50.
In some embodiments, the present disclosure provides a method for detecting corneal dystrophy, the method comprising: (A) amplifying at least two TGFβI gene sequences, including a first TGFβI gene sequence comprising nucleotides encoding amino acid residue 124 and a second TGFβI gene sequence comprising nucleotides encoding amino acid residue 555, from a biological sample from a human subject using a reaction mixture comprising at least a first amplification primer pair and at least a second amplification primer pair; (B) hybridizing a first detection probes of a first detection oligonucleotide probe pair to the first TGFβI gene sequence; (C) hybridizing a second detection probe of a second detection oligonucleotide probe pair to the second TGFβI gene sequence; and (D) detecting one or more mutations in the first TGFβI gene sequence and/or the second TGFβI gene sequence based on a use of at least two detection probe pairs, including the first detection oligonucleotide probe pair and the second detection oligonucleotide probe pair.
In some embodiments, detecting the one or more mutations in the first TGFβI gene sequence and/or the second TGFβI gene sequence includes detecting two or more mutations in the first TGFβI gene sequence and/or the second TGFβI gene sequence and the two or more mutations are separated in the human genome by at least one nucleotide.
In some embodiments, the at least first amplification primer pair comprises a first amplification primer and a second amplification primer, wherein the first amplification primer comprises nucleotide sequence SEQ ID NO:1 and wherein the second amplification primer comprises nucleotide sequence SEQ ID NO:2.
In some embodiments, the at least second amplification primer pair comprises a third amplification primer and a fourth amplification primer, wherein the third amplification primer is represented by nucleotide sequence SEQ ID NO:43, and wherein the fourth amplification primer is represented by nucleotide sequence SEQ ID NO:44.
In some embodiments, 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, the second amplification primer comprises nucleotide sequence SEQ ID NO:2, the second amplification primer pair comprises a third amplification primer and a fourth amplification primer, the third amplification primer comprises nucleotide sequence SEQ ID NO:43, and the fourth amplification primer comprises nucleotide sequence SEQ ID NO:44.
In some embodiments, one of the one or more mutations corresponds to, at amino acid 124, arginine mutated to a cysteine (R124C), arginine mutated to a histidine (R124H), and/or arginine mutated to a leucine (R124L) in the encoded TGFBI protein.
In some embodiments, one of the one or more mutations corresponds to, at amino acid 555, arginine mutated to a tryptophan (R555W) and/or arginine mutated to a glutamine (R555Q) in the encoded TGFBI protein.
In some embodiments, each of the at least two detection oligonucleotide probe pairs individually includes a first detection oligonucleotide probe and a second detection oligonucleotide probe, wherein the first detection oligonucleotide probe and the second detection oligonucleotide probe comprise a nucleotide sequence pair individually selected from the group consisting of SEQ ID NOs:25-26, SEQ ID NOs:25 and 48, SEQ ID NOs:25 and 49, SEQ ID NOs:27-28, SEQ ID NOs:29-30, SEQ ID NOs:31-32, SEQ ID NOs:33-34, SEQ ID NOs:35-36, SEQ ID NOs:37-38, SEQ ID NOs:39-40, SEQ ID NOs:41-42, SEQ ID NOs:45-46 and SEQ ID NOs:46-47, SEQ ID NOs: 45 and 50, and SEQ ID NOs: 47 and 50.
In some embodiments, the first detection oligonucleotide probe pair comprises a first detection oligonucleotide probe and a second detection oligonucleotide probe, wherein the first detection oligonucleotide probe and the second detection oligonucleotide probe respectively comprise nucleotide sequence pair SEQ ID NO:25 and SEQ ID NO:26.
In some embodiments, the second detection oligonucleotide probe pair comprises a third detection oligonucleotide probe and a fourth detection oligonucleotide probe, wherein the third detection oligonucleotide probe and the fourth detection oligonucleotide probe respectively comprise nucleotide sequence pair SEQ ID NO:45 and SEQ ID NO:46.
In some embodiments, the second detection oligonucleotide probe pair comprises a third detection oligonucleotide probe and a fourth detection oligonucleotide probe, wherein the third detection oligonucleotide probe and the fourth detection oligonucleotide probe respectively comprise nucleotide sequence pair SEQ ID NO:47 and SEQ ID NO:46.
In some embodiments, the second detection oligonucleotide probe pair comprises a third detection oligonucleotide probe and a fourth detection oligonucleotide probe, wherein the third detection oligonucleotide probe and the fourth detection oligonucleotide probe respectively comprise nucleotide sequence pair SEQ ID NO:25 and SEQ ID NO:48.
In some embodiments, the second detection oligonucleotide probe pair comprises a third detection oligonucleotide probe and a fourth detection oligonucleotide probe, wherein the third detection oligonucleotide probe and the fourth detection oligonucleotide probe respectively comprise nucleotide sequence pair SEQ ID NO:25 and SEQ ID NO:49.
In some embodiments, the second detection oligonucleotide probe pair comprises a third detection oligonucleotide probe and a fourth detection oligonucleotide probe, wherein the third detection oligonucleotide probe and the fourth detection oligonucleotide probe respectively comprise nucleotide sequence pair SEQ ID NO:45 and SEQ ID NO:50.
In some embodiments, the second detection oligonucleotide probe pair comprises a third detection oligonucleotide probe and a fourth detection oligonucleotide probe, wherein the third detection oligonucleotide probe and the fourth detection oligonucleotide probe respectively comprise nucleotide sequence pair SEQ ID NO:47 and SEQ ID NO:50.
In some embodiments, the first detection probe is coupled with a first label and the second detection probe is coupled to a second label.
In some embodiments, the first label is VIC and the second label is FAM.
In some embodiments, the hybridizing (B) and the hybridizing (C) are performed concurrently in a same solution.
In some embodiments, the hybridizing (B) and the hybridizing (C) are performed concurrently or at different time in a same solution or different solutions.
In some embodiments, the present disclosure provides a reaction mixture for detecting corneal dystrophy in a human subject, the reaction mixture comprising: (A) at least a first amplification primer pair and a second amplification primer pair for amplifying and determining (1) a first TGFβI gene sequence of at least two TGFβI gene sequences that comprises nucleotides encoding amino acid residue 124 from a biological sample from the subject and (2) a second TGFβI gene sequence of the at least two TGFβI gene sequences that comprises nucleotides encoding amino acid residue 555 from a biological sample from the subject; and (B) at least two detection probe pairs, wherein a detection probe in each of the at least two detection probe pairs hybridizes to at least one of the at least two TGFβI gene sequences.
In some embodiments, the first amplification primer pair comprises a first amplification primer and a second amplification primer, the first amplification primer comprises a nucleotide sequence SEQ ID NO:1, and the second amplification primer comprises a nucleotide sequence SEQ ID NO:2.
In some embodiments, the second amplification primer pair comprises a third amplification primer and a fourth amplification primer, the third amplification primer comprises a nucleotide sequence SEQ ID NO:43, and the fourth amplification primer comprises a nucleotide sequence SEQ ID NO:44.
In some embodiments, at least one of the at least two detection probe pairs is used to detect a mutation that corresponds to, at amino acid 124, arginine mutated to a cysteine (R124C), arginine mutated to a histidine (R124H), and/or arginine mutated to a leucine (R124L) in the encoded TGFBI protein.
In some embodiments, at least one of the at least two detection probe pairs is used to detect a mutation that corresponds to, at amino acid 555, arginine mutated to a tryptophan (R555W) and/or arginine mutated to a glutamine (R555Q) in the encoded TGFBI protein.
In some embodiments, respective detection probe pairs of the at least two detection probe pairs individually include a first detection oligonucleotide probe and a second detection oligonucleotide probe, wherein the first detection oligonucleotide probe and the second detection oligonucleotide probe comprise a nucleotide sequence pair individually selected from the group consisting of SEQ ID NOs:25-26, SEQ ID NOs:25 and 48, SEQ ID NOs:25 and 49, SEQ ID NOs:27-28, SEQ ID NOs:29-30, SEQ ID NOs:31-32, SEQ ID NOs:33-34, SEQ ID NOs:35-36, SEQ ID NOs:37-38, SEQ ID NOs:39-40, SEQ ID NOs:41-42, SEQ ID NOs: 45-46 and SEQ ID NOs: 46-47, SEQ ID NOs: 45 and 50, and SEQ ID NOs: 47 and 50.
In some embodiments, one of the at least two detection probe pairs comprises a first detection oligonucleotide probe and a second detection oligonucleotide probe, wherein the first detection oligonucleotide probe and the second detection oligonucleotide probe respectively comprise nucleotide sequence pair SEQ ID NO:25 and SEQ ID NO:26.
In some embodiments, one of the at least two detection probe pairs comprises a first detection oligonucleotide probe and a second detection oligonucleotide probe, wherein the first detection oligonucleotide probe and the second detection oligonucleotide probe respectively comprise nucleotide sequence pair SEQ ID NO:45 and SEQ ID NO:46.
In some embodiments, one of the at least two detection probe pairs comprises a first detection oligonucleotide probe and a second detection oligonucleotide probe, wherein the first detection oligonucleotide probe and the second detection oligonucleotide probe respectively comprise nucleotide sequence pair SEQ ID NO:47 and SEQ ID NO:46.
In some embodiments, one of the at least two detection probe pairs comprises a first detection oligonucleotide probe and a second detection oligonucleotide probe, wherein the first detection oligonucleotide probe and the second detection oligonucleotide probe respectively comprise nucleotide sequence pair SEQ ID NO:25 and SEQ ID NO:48.
In some embodiments, one of the at least two detection probe pairs comprises a first detection oligonucleotide probe and a second detection oligonucleotide probe, wherein the first detection oligonucleotide probe and the second detection oligonucleotide probe respectively comprise nucleotide sequence pair SEQ ID NO:25 and SEQ ID NO:49.
In some embodiments, one of the at least two detection probe pairs comprises a first detection oligonucleotide probe and a second detection oligonucleotide probe, wherein the first detection oligonucleotide probe and the second detection oligonucleotide probe respectively comprise nucleotide sequence pair SEQ ID NO:45 and SEQ ID NO:50.
In some embodiments, one of the at least two detection probe pairs comprises a first detection oligonucleotide probe and a second detection oligonucleotide probe, wherein the first detection oligonucleotide probe and the second detection oligonucleotide probe respectively comprise nucleotide sequence pair SEQ ID NO:47 and SEQ ID NO:50.
In some embodiments, a first detection probe of the at least two detection probes is coupled with a first label and a second detection probe of the at least two detection probes is coupled to a second label.
In some embodiments, the first label is VIC and the second label is FAM.
In some embodiments, the present disclosure provides a reaction mixture for detecting corneal dystrophy in a human subject, the reaction mixture comprising: (A) at least a first amplification primer pair for amplifying and determining a TGFβI gene sequence from a biological sample from the subject; and (B) a set of at least three detection probes, wherein one detection probe of the set of at least three detection probes hybridizes to the TGFβI gene sequence when exposed to the TGFβI gene sequence.
In some embodiments, the TGFβI gene sequence comprises nucleotides encoding amino acid residue 124 from the biological sample from the subject.
In some embodiments, the set of at least three detection probes include at least one detection probe selected from the group consisting of SEQ ID NOs:25-42 and 48-49.
In some embodiments, the set of at least three detection probes include at least two or more detection probes selected from the group consisting of SEQ ID NOs:25-42 and 48-49.
In some embodiments, the set of at least three detection probes include at least three or more detection probes selected from the group consisting of SEQ ID NOs:25-42 and 48-49.
In some embodiments, the set of at least three detection probes include a first detection probe SEQ ID NO:25 and a second detection probe SEQ ID NO:26.
In some embodiments, the set of at least three detection probes include a third detection probe selected from the group consisting of SEQ ID NO:48 and SEQ ID NO:49.
In some embodiments, the set of at least three detection probes include a fourth detection probe that is distinct from the third detection probe and that is selected from the group consisting of SEQ ID NO:48 and SEQ ID NO:49.
In some embodiments, the set of at least three detection probes include two or more detection probes selected from the group consisting of SEQ ID NOs:26 and 48-49.
In some embodiments, the first amplification primer pair comprises a first amplification primer and a second amplification primer, the first amplification primer comprises a nucleotide sequence SEQ ID NO:1, and the second amplification primer comprises a nucleotide sequence SEQ ID NO:2.
In some embodiments, the reaction mixture further comprises: (C) at least a second amplification primer pair for amplifying and determining a second TGFβI gene sequence from the biological sample; and (D) a second set of at least three detection probes, wherein one detection probe of the second set of at least three detection probes hybridizes the second TGFβI gene sequence when exposed to the second TGFβI gene sequence.
In some embodiments, the second TGFβI gene sequence comprises nucleotides encoding amino acid residue 555 from the biological sample from the subject.
In some embodiments, the second set of at least three detection probes include at least one detection probe selected from the group consisting of SEQ ID NOs: 45-47 and 50.
In some embodiments, the second set of at least three detection probes include at least two or more detection probes selected from the group consisting of SEQ ID NOs: 45-47 and 50.
In some embodiments, the second set of at least three detection probes include at least three or more detection probes selected from the group consisting of SEQ ID NOs: 45-47 and 50.
In some embodiments, the second set of at least three detection probes include a first detection probe that is SEQ ID NO:45 or SEQ ID NO:47, a second detection probe that is SEQ ID NO:46, and a third detection probe that is SEQ ID NO:50.
In some embodiments, the second amplification primer pair comprises a third amplification primer and a fourth amplification primer, the third amplification primer comprises a nucleotide sequence SEQ ID NO:43, and the fourth amplification primer comprises a nucleotide sequence SEQ ID NO:44.
In some embodiments, the TGFβI gene sequence comprises nucleotides encoding amino acid residue 555 from the biological sample from the subject.
In some embodiments, the set of at least three detection probes include at least one detection probe selected from the group consisting of SEQ ID NOs:45-47 and 50.
In some embodiments, the set of at least three detection probes include at least two or more detection probes selected from the group consisting of SEQ ID NOs: 45-47 and 50.
In some embodiments, the set of at least three detection probes include at least three or more detection probes selected from the group consisting of SEQ ID NOs: 45-47 and 50.
In some embodiments, the set of at least three detection probes include a first detection probe that is SEQ ID NO:45 or SEQ ID NO:47, a second detection probe that is SEQ ID NO:46, and a third detection probe that is SEQ ID NO:50.
In some embodiments, the present disclosure provides a method for detecting corneal dystrophy comprising: (A-1) amplifying a first TGFβI gene sequence from a biological sample from a human subject using a reaction mixture comprising at least a first amplification primer pair and a set of at least three detection probes; (B-1) hybridizing a first detection probe of the set of at least three detection probes to the first TGFβI gene sequence; and (C-1) detecting a mutation in the first TGFβI gene sequence based on (i) the hybridization of the first detection probe of the set of at least three detection probes to the first TGFβI gene sequence and (ii) the failure of a second and a third detection probe of the set of at least three detection probes to hybridize to first TGFβI gene sequence.
In some embodiments, the method further comprises: (A-2) amplifying a second TGFβI gene sequence from the biological sample using the same reaction mixture, wherein the reaction mixture comprises at least a second amplification primer pair and a second set of at least three detection probes; (B-2) hybridizing a first detection probe of the second set of at least three detection probes to the second TGFβI gene sequence; and (C-2) detecting a mutation in the second TGFβI gene sequence based on (i) the hybridization of the first detection probe of the second set of at least three detection probes to the first TGFβI gene sequence and (ii) and (ii) the failure of a second detection probe and a third detection probe of the second set of at least three detection probes to hybridize to the second TGFβI gene sequence.
In some embodiments, the amplifying (A-1), the amplifying (A-2), the hybridizing (B-1), the hybridizing (B-2), the detecting (C-1), and the detecting (C-2) are performed with a same aliquot of the biological sample.
In some embodiments, the amplifying the first TGFβI gene sequence (A-1) and the amplifying the second TGFβI gene sequence (A-2) are performed concurrently.
In some embodiments, the hybridizing (B-1) and the hybridizing (B-2) are performed concurrently.
In some embodiments, the detecting (C-1) and the detecting (C-2) are performed concurrently.
In some embodiments, the reaction mixture has some of the features described above. For brevity, such details are not repeated herein.
In some embodiments, the present disclosure provides use of a reaction mixture, as recited in any above, for predicting the risk of complication following laser eye surgery in a subject through a detection of heterozygous corneal dystrophy in the human subject.
In some embodiments, the laser eye surgery comprises one of Lasik and Excimer laser surgery.
In some embodiments, the present disclosure provides a method for detecting a genomic mutation associated with corneal dystrophy in a sample from a human subject, the method comprising: (A) providing epithelial cells of a human subject adhered to a tip of a substrate; (B) agitating the tip of the substrate in a lysis solution that lyses cells adhered to the substrate; (C) removing the substrate from the lysis solution upon completion of the agitating (B); (D) incubating the lysis solution after the removing (C); (E) isolating genomic DNA from the lysis solution to form a gDNA solution; and (F) determining an identity of at least a nucleotide present in the TGFβI gene using at least a first primer pair, a set of at least three detection probes, and the gDNA solution by concurrently exposing the gDNA solution to the at least three detection probes, wherein: the at least a nucleotide is located at a particular position of the TGFβI gene corresponding to a single nucleotide polymorphism (SNPs) associated with corneal dystrophy, and each of at least two detection probes of the set of the at least three detection probes is configured to detect a different mutation at the particular position of the TGFβI gene.
In some embodiments, the method further comprises: (G) determining an identity of at least a second nucleotide present in the TGFβI gene using at least a second primer pair, a second set of at least three detection probes, and the gDNA solution by concurrently exposing the gDNA solution to the set of at least three detection probes and the second set of at least three detection probes, wherein: the at least a second nucleotide is located at a second particular position, independent of the position of the at least a nucleotide, of the TGFβI gene corresponding to a second single nucleotide polymorphism (SNPs) associated with corneal dystrophy, and at least two detection probes of the second set of the at least three detection probes are configured to detect a respective mutation at the second particular position of the TGFβI gene.
In some embodiments, the determining (F) and the determining (G) are performed concurrently.
In some embodiments, the determining (F) and the determining (G) are performed using the same gDNA solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B illustrate an improved method 100 for the detection of genomic alleles associated with disease, according to some embodiments.

FIG. 2 provides a list of sequences for forward and reverse PCR primer pairs (SEQ ID NOs:1-24) useful for real-time PCR detection of a single nucleotide polymorphism associated with Avellino corneal dystrophy, in accordance with some embodiments.

FIG. 3 provides a list of sequences for wild type and mutant detection probe pairs (SEQ ID NOs:25-42) useful for real-time PCR detection of a single nucleotide polymorphism associated with Avellino corneal dystrophy, in accordance with some embodiments.

FIG. 4 provides a list of the probe sequences and primers used in the allele detection experiments shown in FIGS. 5 through 8 .

FIGS. 5A, 5B, and 5C provide experimental data regarding detection of the R124C, R124H, R124L, R555W and R555Q mutants using the probes described in FIG. 4 . FIG. 5A provides an allelic discrimination plot run (left-hand panel) using the indicated reagent mixture and indicated cycling conditions (right-hand panel). FIG. 5B provides the Real-Time PCR plots for the various mutants compared to a normal control. FIG. 5C provides the Real-Time PCR plots for the various mutants compared to a homozygous control.

FIGS. 6A, 6B, and 6C provide experimental data regarding detection of the R124C, R124H, R124L, R555W and R555Q mutants using the probes described in FIG. 4 . FIG. 6A provides an allelic discrimination plot run (left-hand panel) using the indicated reagent mixture and indicated cycling conditions (right-hand panel). FIG. 6B provides the Real-Time PCR plots for the various mutants compared to a normal control. FIG. 6C provides the Real-Time PCR plots for the various mutants compared to a homozygous control.

FIG. 7 provides experimental data regarding detection of the R124C, R124H, R124L, R555W and R555Q mutants using the probes described in FIG. 4 . FIG. 7 provides an allelic discrimination plot (left-hand panel) run using the indicated reagent mixture and indicated cycling conditions (right-hand panel).

FIG. 8 provides experimental data regarding detection of the R124C, R124H, R124L, R555W and R555Q mutants using the probes described in FIG. 4 . In particular, FIG. 8 provides an allelic discrimination plot run using the indicated reagent mixture and indicated cycling conditions (right-hand panel). The designation “B” in the allelic discrimination plot (left-hand panel) indicates samples were pre-treated with lysis buffer prior to performing the Real-Time PCR assay. The designation “DW-A” in the allelic discrimination plot (left-hand panel) indicates samples were pre-treated with distilled water prior to performing the Real-Time PCR assay. The circles surrounding the different sample dots show the two matched samples, “B” and “DW-A” respectively, for each of the alleles detected (see, the left-hand panel); there are two dots within each circle, one for sample “B” and one for sample “DW-A”.

DETAILED DESCRIPTION

I. Introduction
The detection of disease-related SNPs is an increasingly more important tool for the diagnosis and prognosis of various medical conditions. For example, the presence of a single nucleotide change in exon 4 of the TGFβI gene is strongly associated with Avellino corneal dystrophy. It was found that individuals heterozygous for this SNP are at high risk for vision loss following LASIK surgery. While LASIK is a medical procedure that greatly improves many people's quality of life, for individuals carrying the G/A TGFβI SNP, it commonly causes a gradual vision impairment over a four to eighteen month period, which may lead to loss of vision. The vision impairment may occur in a longer or shorter period of time. Fortunately, screening can be performed to identify individuals carrying the mutation who should avoid having the LASIK procedure.
The present disclosure is based at least in part on the discovery of methods that improve sample isolation, preparation, and analysis. In some embodiments, methods are provided which allow for the re-use of patient samples, for example, when an assay fails or additional follow-up testing needs to be performed. In some embodiments, these improved methods include gently swirling a substrate (e.g., a rayon-tipped or cotton-tipped applicator) carrying cells sloughed-off the buccal membrane of the patient in a lysis solution at room temperature for 30-45 seconds (rather than extended incubation for 20 minutes at elevated temperature). The lysis solution is then incubated at 45° C. for 30 minutes to improve lysis and increase the yield of genomic sample. Advantageously, the rayon-tipped or cotton-tipped applicator can then be stored (e.g., frozen or refrigerated) for re-isolation of genomic DNA used for re-testing.
In some embodiments, the improvements provided herein are provided through the use of lower amounts of genomic DNA template for the real-time PCR detection assays. In some embodiments, this is achieved by increasing the number of real-time PCR cycles performed (e.g., at about 40 cycles) and/or by using 3 second denaturation cycle times at 95° C. Advantageously, because the amount of sample required is reduced by these methods, so too are the requirements for the real-time PCR reagents. Because many reagents used in diagnostic assays are proprietary, the reagents can be expensive. Reducing the amount of reagent used can also significantly reduce the costs associated with the reagent.
It is contemplated that all combinations of specific conditions (e.g., sample handling, incubation temperature, reaction volumes, reaction cycle numbers, reaction cycle times, reaction cycle temperatures) for performing each of these individual steps can be used to perform the methods described herein for detecting disease-related SNPs, such as the Avellino corneal dystrophy-related SNP found in exon 4 of the TGFβI gene.
II. Select Definitions
The term “invention” or “present invention” as used herein is not meant to be limiting to any one specific embodiment of the invention but applies generally to any and all embodiments of the invention as described in the claims and specification.
As used herein, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure.
As used herein, the term “polymorphism” and variants thereof refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. The terms “genetic mutation” or “genetic variation” and variants thereof include polymorphisms.
As used herein the term “single nucleotide polymorphism” (“SNP”) and variants thereof refers to a site of one nucleotide that varies between alleles. A single nucleotide polymorphism (SNP) is a single base change or point mutation but also includes the so-called “indel” mutations (insertions or deletions of a nucleotide), resulting in genetic variation between individuals. SNPs, which make up about 90% of all human genetic variation, occur every 100 to 300 bases along the 3-billion-base human genome. However, SNPs can occur much more frequently in other organisms like viruses. SNPs can occur in coding or non-coding regions of the genome. A SNP in the coding region may or may not change the amino acid sequence of a protein product. A SNP in a non-coding region can alter promoters or processing sites and may affect gene transcription and/or processing. Knowledge of whether an individual has particular SNPs in a genomic region of interest may provide sufficient information to develop diagnostic, preventive and therapeutic applications for a variety of diseases. In some embodiments, the present disclosure relates to the detection of a guanine-to-adenine SNP located in exon 4 of the TGFβI gene associated with Avellino corneal dystrophy.
The term “primer” and variants thereof refers to an oligonucleotide that acts as a point of initiation of DNA synthesis in a PCR reaction. A primer is usually about 15 to about 35 nucleotides in length and hybridizes to a region complementary to the target sequence.
The term “probe” and variants thereof (e.g., detection probe) refers to an oligonucleotide that hybridizes to a target nucleic acid in a PCR reaction. Target sequence refers to a region of nucleic acid that is to be analyzed and comprises the polymorphic site of interest.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, various embodiments of methods and materials are specifically described herein.
III. Sample Preparation
In some embodiments, the disclosure provides improved methods for isolating genomic samples used in real-time PCR single nucleotide polymorphism detection assays. In some embodiments, the improved method 100 uses a combination of steps outlined in FIG. 1 .
In some embodiments, the method includes providing a sample of cells from a subject. In some embodiments, the cells are collected by contacting a cellular surface of a patient with a substrate capable of reversibly immobilizing the cells onto a substrate.
The disclosed methods are applicable to a variety of cell types obtained from a variety of samples. In some embodiments, the cell type for use with the disclosed methods include but is not limited to epithelial cells, endothelial cells, connective tissue cells, skeletal muscle cells, endocrine cells, cardiac cells, urinary cells, melanocytes, keratinocytes, blood cells, white blood cells, buffy coat, hair cells (including, e.g., hair root cells) and/or salival cells. In some embodiments, the cells are epithelial cells. In some embodiments, the cells are subcapsular-perivascular (epithelial type 1); pale (epithelial type 2); intermediate (epithelial type 3); dark (epithelial type 4); undifferentiated (epithelial type 5); and large-medullary (epithelial type 6). In some embodiments, the cells are buccal epithelial cells (e.g., epithelial cells collected using a buccal swap). In some embodiments, the sample of cells used in the disclosed methods include any combination of the above identified cell types.
In some embodiments, the method includes providing (102) a sample of cells from a subject. In some embodiments, the cells provided are buccal epithelial cells.
The cell sample is collected by any of a variety of methods which allow for reversible binding of the subjects cells to the substrate. In some embodiments, the substrate is employed in a physical interaction with the sample containing the subject's cells in order to reversibly bind the cells to the substrate. In some embodiments, the substrate is employed in a physical interaction with the body of the subject directly in order to reversibly bind the cells to the substrate. In some embodiments, the sample is a buccal cell sample and the sample of buccal cells is collected by contacting a buccal membrane of the subject (e.g., the inside of their cheek) with a substrate capable of reversibly immobilizing cells that are dislodged from the membrane. In such embodiments, the swab is rubbed against the inside of the subject's cheek with a force equivalent to brushing a person's teeth (e.g., a light amount of force or pressure). Any method which would allow the subject's cells to be reversibly bound to the substrate is contemplated for use with the disclosed methods.
In some embodiments, the sample is advantageously collected in a non-invasive manner and as such sample collection is accomplished anywhere and by almost anyone. For example, in some embodiments the sample is collected at a physician's office, at a subject's home, or at a facility where LASIK surgery is performed or to be performed. In some embodiments the patient, the patient's doctor, nurses or a physician's assistant or other clinical personnel collects the sample.
In some embodiments the substrate is made of any of a variety of materials to which cells are reversibly bound. Exemplary substrates include those made of rayon, cotton, silica, an elastomer, a shellac, amber, a natural or synthetic rubber, cellulose, BAKELITE, NYLON, a polystyrene, a polyethylene, a polypropylene, a polyacrylonitrile, or other materials or combinations thereof. In some embodiments, the substrate is a swab having a rayon tip or a cotton tip.
The tip of the substrate (e.g., the tip of the rayon swab or cotton swab) is then agitated in a lysis solution from about 10 seconds to 60 seconds (1 minute), or about 20 seconds to 60 seconds, about 20 seconds to about 45 seconds, or about 20 seconds to about 30 seconds, about 15 seconds to about 60 seconds, about 15 seconds to about 45 seconds, or about 15 seconds to about 30 seconds, about 10 seconds to about 60 seconds, about 10 seconds to about 45 seconds, or about 10 seconds to about 30 seconds, about 10 seconds to about 15 seconds or about 10 seconds to about 20 seconds. In some embodiments, the agitation occurs for about 60 seconds or about 1 minute. In some embodiments, the agitation occurs for less than a minute (e.g., less than 60 seconds). In some embodiments, the agitation occurs for no more than 15 seconds, 20 seconds, 30 seconds, 45 seconds, 60 seconds, 90 seconds, 120 seconds or more. In some embodiments, the agitation occurs for no more than 45 seconds. In some embodiments, the agitation occurs for no more than 30 seconds. In some embodiments, the agitation occurs for no more than 20 seconds. In some embodiments, the agitation occurs for no more than 15 seconds.
In some embodiments, agitation includes any movement of the substrate in the lysis solution. In some embodiments, the tip of the substrate (e.g., the tip of the rayon swab or cotton swab) is moved gently in the lysis solution, such that a plurality of buccal cells remains affixed to the substrate for isolation at a later time and/or subsequent time. Such movement in the lysis solution includes swirling motions, side to side motions, up and down motions and/or dipping motions, or any other movement of the substrate in the lysis solutions that results in a plurality of buccal cell remain affixed to the tip while allowing for some buccal cells to be dispersed into the lysis solution.
In some embodiments, the agitation step is performed at room temperature, for instance, temperatures between about 15° C. and about 30° C., about 18° C. and about 28° C., about 18° C. and about 25° C. or about 20° C. and about 25° C.
After agitation, the substrate (e.g., a swab with a rayon tip or cotton tip) is removed and, in some embodiments, stored for use later, in case re-testing or further (e.g., different or additional) testing is needed. In some embodiments, the substrate (e.g., buccal swab with a rayon tip or cotton tip) is placed in a container and stored frozen. In some embodiments, the substrate (e.g., buccal swab with a rayon tip or cotton tip) is refrigerated. In some embodiments, the substrate is stored at any of a variety of temperatures and for any of a variety of times while still remaining useful for one or more additional extractions.
In some embodiments, the substrate containing the sample is stored for 0 weeks, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks or more. In some embodiments, the substrate containing the sample is stored for and/or is capable of being stored for 0 weeks to 12 weeks, 1 week to 12 weeks, 2 weeks to 12 weeks, 3 weeks to 12 weeks, 4 weeks to 12 weeks, 5 weeks to 12 weeks, 6 weeks to 12 weeks, 7 weeks to 12 weeks, 8 weeks to 12 weeks, 9 weeks, 10 weeks to 12 weeks, or 11 weeks to 12 weeks. In some embodiments, the substrate containing the sample is stored for 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 30, or 36 months or more. In some embodiments, the substrate containing the sample is stored for 1 month to 36 months, 2 months to 36 months, 3 months to 36 months, 4 months to 36 months, 5 months to 36 months, 6 months to 36 months, 7 months to 36 months, 8 months to 36 months, 9 months to 36 months, 10 months to 36 months, 12 months to 36 months, 14 months to 36 months, 16 months to 36 months, 18 months to 36 months. In some embodiments, the substrate containing the sample is stored for 1 month to 30 months, 2 months to 30 months, 3 months to 30 months, 4 months to 30 months, 5 months to 30 months, 6 months to 30 months, 7 months to 30 months, 8 months to 30 months, 9 months to 30 months, 10 months to 30 months, 12 months to 30 months, 14 months to 30 months, 16 months to 30 months or 18 months to 30 months. In some embodiments, the substrate containing the sample is stored for 1 month to 24 months, 2 months to 24 months, 3 months to 24 months, 4 months to 24 months, 5 months to 24 months, 6 months to 24 months, 7 months to 24 months, 8 months to 24 months, 9 months to 24 months, 10 months to 24 months, 12 months to 24 months, 14 months to 24 months, 16 months to 24 months, 18 months to 24 months. In some embodiments, the substrate containing the sample is stored for 1 month to 22 months, 2 months to 22 months, 3 months to 22 months, 4 months to 22 months, 5 months to 22 months, 6 months to 22 months, 7 months to 22 months, 8 months to 22 months, 9 months to 22 months, 10 months to 22 months, 12 months to 22 months, 14 months to 22 months, 16 months to 22 months, 18 months to 22 months. In some embodiments, the substrate containing the sample is stored for 1 month to 20 months, 2 months to 20 months, 3 months to 20 months, 4 months to 20 months, 5 months to 20 months, 6 months to 20 months, 7 months to 20 months, 8 to 20 months, 9 to 20 months, 10 months to 20 months, 12 months to 20 months, 14 months to 20 months, 16 months to 20 months, 18 months to 20 months. In some embodiments, the substrate containing the sample is stored for 1 month to 18 months, 2 months to 18 months, 3 months to 18 months, 4 months to 18 months, 5 months to 18 months, 6 months to 18 months, 7 months to 18 months, 8 months to 18 months, 9 months to 18 months, 10 months to 18 months, 12 months to 18 months, 14 months to 18 months, 16 months to 18 months or 17 months to 18 months. In some embodiments, the substrate containing the sample is stored for 1 month to 12 months, 2 months to 12 months, 3 months to 12 months, 4 months to 12 months, 5 months to 12 months, 6 months to 12 months, 7 months to 12 months, 8 months to 12 months, 9 months to 12 months, 10 months to 12 months or 11 months to 12 months.
In some embodiments, the substrate containing the sample is stored at about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., or about 8° C. In some embodiments, the substrate containing the sample is stored at about 2° C. to about 8° C., about 3° C. to about 8° C., about 4° C. to about 8° C., about 5° C. to about 8° C., about 6° C. to about 8° C. or about 7° C. to about 8° C. In some embodiments, the substrate containing the sample is stored at about −25° C., about −24° C., about −23° C., about −22° C., about −21° C., about −20° C., about −19° C., about −18° C., about −17° C., about −16° C. or about −15° C. In some embodiments, the substrate containing the sample is stored at about −25° C. to about −15° C., about −22° C. to about −17° C., about −20° C. to about −15° C. or about −25° C. to about −20° C. In some embodiments, the substrate containing the sample is stored at about −90° C., about −89° C., about −88° C., about −87° C., about −86° C., about −85° C., about −84° C., about −83° C., about −82° C., about −81° C., about −80° C., about −79° C., about −78° C., about −77° C., about −76° C., about −75° C., about −74° C., about −73° C., about −72° C., about −71° C., about −70° C., about −69° C., about −68° C., about −67° C., about −66° C. or about −65° C. In some embodiments, the substrate containing the sample is stored at about −90° C. to about −65° C., about −85° C. to about −65° C., about −80° C. to about −65° C., about −75° C. to about −65° C. or about −70° C. to about −65° C. In some embodiments, the substrate containing the sample is stored at −90° C. to −65° C.
In some embodiments, the substrate containing the sample is freeze-thawed one or more times (e.g., after being frozen, the substrate containing the sample is thawed, used according to the present methods and re-frozen) and or used in the present methods. In some embodiments, the substrate containing the sample is freeze-thawed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times. In some embodiments, the substrate containing the sample is used in the present methods 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times. In some embodiments, the substrate containing the sample is freeze-thawed 1 to 20 times, 2 to 20 times, 3 to 20 times, 4 to 30 times, 5 to 20 times, 6 to 20 times, 7 to 20 times, 8 to 20 times, 9 to 20 times, 10 to 20 times, 11 to 20 times, 12 to 20 times, 13 to 20 times, 14 to 20 times, 15 to 20 times, 16 to 20 times, 17 to 20 times, 18 to 20 times, 19 to 20 times, 5 to 15 times, 5 to 10 times, 1 to 10 times or 1 to 5 times. In some embodiments, the substrate containing the sample is used in the present methods 1 to 20 times, 2 to 20 times, 3 to 20 times, 4 to 30 times, 5 to 20 times, 6 to 20 times, 7 to 20 times, 8 to 20 times, 9 to 20 times, 10 to 20 times, 11 to 20 times, 12 to 20 times, 13 to 20 times, 14 to 20 times, 15 to 20 times, 16 to 20 times, 17 to 20 times, 18 to 20 times, 19 to 20 times, 5 to 15 times, 5 to 10 times, 1 to 10 times, 1 to 5 times, 1 to 4 times, 1 to 3 times or 1 to 2 times.
In some embodiments, the substrate containing the sample is stored for 1 week at room temperature or about 15° C. to about 30° C. In some embodiments, the sample is stored for about 1, 2 or 3 weeks at about 2° C. to about 8° C. or about 4° C. In some embodiments, the substrate containing the sample is stored for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months at about −25° C. to about −15° C. or about −20° C. In some embodiments, the substrate containing the sample is stored for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months at about −90° C. to about −65° C. or about −80° C.
Advantageously and surprisingly, it was found that the reduced number of cells extracted from the substrate is countered by increased extraction of nucleic acids from individual cells. In some embodiments, increased extraction is accomplished by incubating the cells for a longer time as compared to standard practices, incubating the cells at an elevated temperature as compared to standard practices, or a combination of both.
In some embodiments, the increased extraction of nucleic acids of cells is accomplished by performing the extraction incubation for an increased or longer period of time as compared to standard practice. In some embodiments, the extraction incubation is performed for about 45 minutes, e.g., 45±5, 45±10, 45±15, or 45±20 minutes. In some embodiments, the extraction incubation is performed for about 25 minutes to about 65 minutes, about 30 minutes to about 60 minutes, about 35 minutes to about 55 minutes, about 45 minutes to about 65 minutes, about 45 minutes to about 55 minutes, or about 40 minutes to about 50 minutes. In some embodiments, the extraction incubation time is about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes or about 65 minutes.
In some embodiments, the increased the extraction of nucleic acids of cells is accomplished by performing the extraction incubation at an increased or higher temperature as compared to standard practice. In some embodiments, the extraction incubation is performed at about 45° C., e.g., 45±2° C., 45±5° C., or 45±10° C. In some embodiments, the extraction incubation temperature is about 35° C. to about 55° C., about 40° C. to about 50° C. or about 43° C. to about 47° C. In some embodiments, the extraction temperature is about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., about 50° C., about 51° C., about 52° C., about 53° C., about 54° C. or about 55° C. In some embodiments, more than one extraction temperature is used. For example in some embodiments, standard temperatures are used for a portion of the extraction and an elevated temperature is used for another portion of the extraction.
In some embodiments, substantially small numbers of cells are released from the substrate for subsequent lysis according to the present systems and methods. In some embodiments, at least 1 cell, at least 2 cells, at least 5 cells, at least 10 cells, at least 15 cells, at least 20 cells, at least 50 cells, at least 75 cells, at least 100 cells, at least 125 cells, at least 150 cells, at least 175 cells, at least 200 cells, at least 250 cells, at least 300 cells, at least 350 cells, at least 400 cells, at least 450 cells, at least 500 cells or more are released from the substrate during agitation.
In some embodiments, about 1 ng/μL to about 50 ng/μL, about 1 ng/μL to about 40 ng/μL, about 1 ng/μL to about 30 ng/μL, about 1 ng/μL to about 20 ng/μL, about 1 ng/μL to about 10 ng/μL, about 1 ng/μL to about 5 ng/μL, about 1 ng/μL to about 4 ng/μL, about 1 ng/μL to about 3 ng/μL or about 1 ng/μL to about 2 ng/μL of nucleic acid with a purity of about 0.55 to 2.00, about 0.6 to about 2.00, about 0.7 to about 2.00 about 0.8 to about 2.00, about 0.9 to about 2.00, about 1.0 to about 2.00 about 1.1 to about 2.00, about 1.2 to about 2.00, about 1.3 to about 2.00, about 1.4 to about 2.00, about 1.5 to about 2.00 about 1.6 to about 2.00 about 1.7 to about 2.00 about 1.8 to about 2.00 or about 1.9 to about 2.00 is employed (obtained) from a single subject with the described methods. In some embodiments, 1 ng/μL to 50 ng/μL with a purity of about 0.55 to 2.00 is employed (obtained) from a single subject with the described methods.
IV. Lysis Solutions
A variety of lysis solutions have been described and are known to those of skill in the art. Any of these well known lysis solutions can be employed with the present methods in order to isolate nucleic acids from a sample. Exemplary lysis solutions include those commercially available, such as those sold by INVITROGEN®, QIAGEN®, LIFE TECHNOLOGIES® and other manufacturers, as well as those which can be generated by one of skill in a laboratory setting. Lysis buffers have also been well described and a variety of lysis buffers can find use with the disclosed methods, including for example those described in Molecular Cloning (three volume set, Cold Spring Harbor Laboratory Press, 2012) and Current Protocols (Genetics and Genomics; Molecular Biology; 2003-2013), both of which are incorporated herein by reference for all purposes.
Cell lysis is a commonly practiced method for the recovery of nucleic acids from within cells. In many cases, the cells are contacted with a lysis solution, commonly an alkaline solution comprising a detergent, or a solution of a lysis enzyme. Such lysis solutions typically contain salts, detergents and buffering agents, as well as other agents that one of skill would understand to use. After full and/or partial lysis, the nucleic acids are recovered from the lysis solution.
In some embodiments, cells are resuspended in an aqueous buffer, with a pH in the range of from about pH 4 to about 10, about 5 to about 9, about 6 to about 8 or about 7 to about 9.
In some embodiments, the buffer salt concentration is from about 10 mM to about 200 mM, about 10 mM to about 100 mM or about 20 mM to about 80 mM.
In some embodiments, the buffer further comprises chelating agents such as ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA).
In some embodiments, the lysis solution further comprises other compounds to assist with nucleic acid release from cells such as polyols, including for example but not limited to sucrose, as well as sugar alcohols such as maltitol, sorbitol, xylitol, erythritol, and/or isomalt. In some embodiments, polyols are in the range of from about 2% to about 15% w/w, or about 5% to about 15% w/w or about 5% to about 10% w/w.
In some embodiments, the lysis solutions further comprises surfactants, such as for example but not limited to Triton X-100, SDS, CTAB, X-114, CHAPS, DOC, and/or NP-40. In some embodiments such surfactants are in the range of from about 1% to about 5% w/w, about 1% to about 4% w/w, or about 1% to about 3% w/w.
In embodiments, the lysis solution further comprises chaotropes, such as for example but not limited to urea, sodium dodecyl sulfate and/or thiourea. In some embodiments, the chaotrope is used at a concentration in the range of from about 0.5 M to 8 M, about 1 M to about 6 M, about 2 M to about 6 M or about 1 M to 3 M.
In some embodiments, the lysis solution further comprises one or more additional lysis reagents and such lysis reagents are well known in the art. In some embodiments, such lysis reagents include cell wall lytic enzymes, such as for example but not limited to lysozyme. In some embodiments, lysis reagents comprise alkaline detergent solutions, such as 0.1 aqueous sodium hydroxide containing 0.5% sodium dodecyl sulphate.
In some embodiments, the lysis solution further comprises aqueous sugar solutions, such as sucrose solution and chelating agents such as EDTA, for example the STET buffer. In certain embodiments, the lysis reagent is prepared by mixing the cell suspension with an equal volume of lysis solution having twice the desired concentration (for example 0.2 sodium hydroxide, 1.0% sodium dodecyl sulphate).
In some embodiments, after the desired extent of lysis has been achieved, the mixture comprising lysis solution and lysed cells is contacted with a neutralizing or quenching reagent to adjust the conditions such that the lysis reagent does not adversely affect the desired product. In some embodiments, the pH is adjusted to a pH of from about 5 to about 9, about 6 to about 8, about 5 to about 7, about 6 to about 7 or about 6.5 to 7.5 to minimize and/or prevent degradation of the cell contents, including for example but not limited to the nucleic acids. In some embodiments, when the lysis reagent comprises an alkaline solution, the neutralizing reagent comprises an acidic buffer, for example an alkali metal acetate/acetic acid buffer. In some embodiments, lysis conditions, such as temperature and composition of the lysis reagent are chosen such that lysis is substantially completed while minimizing degradation of the desired product, including for example but not limited to nucleic acids.
In some embodiments, a first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth or twentieth lysis solution is employed with the methods. In some embodiments, the volume of lysis buffer used is about 10 μL, about 20 μL, about 30 μL, about 40 μL, about 50 μL, about 60 μL, about 70 μL, about 80 μL, about 90 μL, about 100 μL, about 120 μL, about 130 μL, about 140 μL, about 150 μL, 160 μL, about 170 μL, about 180 μL, about 190 μL, about 200 μL, about 220 μL, about 230 μL, about 240 μL, about 250 μL, about 260 μL, about 270 μL, about 280 μL, about 290 μL, about 300 μL, about 320 μL, about 330 μL, about 340 μL, about 350 μL, about 360 μL, about 370 μL, about 380 μL, about 390 μL, about 400 μL, about 450 μL, about 500 μL, about 550 μL, about 600 μL, about 650 μL, about 700 μL, about 750 μL, about 800 μL, about 850 μL, 900 μL, 950 μL, 1000 μL, 1500 μL or 2000 μL. In some embodiments, the lysis buffer is between about 10 μL and about 1000 μL, about 10 μL and about 800 μL, about 10 μL and about 600 μL, about 10 μL and about 400 μL, about 20 μL and about 400 μL, about 50 μL and about 300 μL, about 50 μL and about 200 μL, about 50 μL and about 400 μL, about 100 μL and about 400 μL, about 10 μL and about 300 μL or about 100 μL and about 200 μL.
Any combination of the above can be employed by one of skill, as well as combined with other known and routine methods, and such combinations are contemplated by the present invention.
V. Purification of Nucleic Acids from Lysis Buffer
In some embodiments, the nucleic acids, including for example but not limited to genomic DNA, are isolated from lysis buffer prior to performing subsequent analysis. In some embodiments, the nucleic acids are isolated from the lysis buffer prior to the performance of additional analyses, such as for example but not limited to real-time PCR analyses. Any of a variety of methods useful in the isolation of small quantities of nucleic acids are used by various embodiments of the disclosed methods. These include but are not limited to precipitation, gel filtration, density gradients and solid phase binding. Such methods have also been described in for example, Molecular Cloning (three volume set, Cold Spring Harbor Laboratory Press, 2012) and Current Protocols (Genetics and Genomics; Molecular Biology; 2003-2013), incorporated herein by reference for all purposes.
Nucleic Acid precipitation is a well know method for isolation that is known by those of skill in the art. A variety of solid phase binding methods are also known in the art including but not limited to solid phase binding methods that make use of solid phases in the form of beads (e.g., silica, magnetic), columns, membranes or any of a variety other physical forms known in the art. In some embodiments, solid phases used in the disclosed methods reversibly bind nucleic acids. Examples of such solid phases include so-called “mixed-bed” solid phases are mixtures of at least two different solid phases, each of which has a capacity to nucleic acids under different solution conditions, and the ability and/or capacity to release the nucleic acid under different conditions; such as those described in US Patent Application No. 2002/0001812, incorporated by reference herein in its entirety for all purposes. Solid phase affinity for nucleic acids according to the disclosed methods can be through any one of a number of means typically used to bind a solute to a substrate. Examples of such means include but are not limited to, ionic interactions (e.g., anion-exchange chromatography) and hydrophobic interactions (e.g., reversed-phase chromatography), pH differentials and changes, salt differentials and changes (e.g., concentration changes, use of chaotropic salts/agents). Exemplary pH based solid phases include but are not limited to those used in the INVITROGEN ChargeSwitch Normalized Buccal Kit magnetic beads, to which bind nucleic acids at low pH (<6.5) and releases nucleic acids at high pH (>8.5) and mono-amino-N-aminoethyl (MANAE) which binds nucleic acids at a pH of less than 7.5 and release nucleic acids at a pH of greater than 8. Exemplary ion exchange based substrates include but are not limited to DEA-SEPHAROSE™, Q-SEPHAROSE™, and DEAE-SEPHADEX™ from PHARMACIA (Piscataway, N.J.), DOWEX® I from The Dow Chemical Company (Midland, Mich.), AMBERLITE® from Rohm & Haas (Philadelphia, Pa.), DUOLITE® from Duolite International, In. (Cleveland, Ohio), DIALON TI and DIALON TII.
Any individual method is contemplated for use alone or in combination with other methods, and such useful combination are well known and appreciated by those of skill in the art.
VI. Nucleic Acid Analyses
The disclosed methods are used to isolate nucleic acids, such as genomic DNA (gDNA) for a variety of nucleic acid analyses, including genomic analyses. In some embodiments, such analysis includes detection of variety of genetic mutations, which include but are not limited to one or more deletions, insertions, transitions and transversions. In some embodiments, the mutation is a single-nucleotide polymorphism (SNP).
A variety of methods for analyzing such isolated nucleic acids, for example but not limited to genomic DNA (gDNA) are known in the art and include PCR methods, such as real-time PCR analysis, microarray analysis, hybridization analysis and nucleic acid sequence analysis, as well as a variety of other methods where nucleic acid compositions are analyzed and which are known to those of skill in the art. See, for example, Molecular Cloning (three volume set, Cold Spring Harbor Laboratory Press, 2012) and Current Protocols (Genetics and Genomics; Molecular Biology; 2003-2013).
a. Real-Time PCR
For the design of Real-Time PCR assays, several parts are coordinated, including the DNA fragment that is flanked by the two primers and subsequently amplified, often referred to as the amplicon, the two primers and the detection probe or probes to be used.
Real-time PCR relies on the visual emission of fluorescent dyes conjugated to short polynucleotides (termed “detection probes”) that associate with genomic alleles in a sequence-specific fashion. Real-time PCR probes differing by a single nucleotide can be differentiated in a real-time PCR assay by the conjugation and detection of probes that fluoresce at different wavelengths. Real-Time PCR finds use in detection applications (diagnostic applications), quantification applications and genotyping applications.
Several related methods for performing real-time PCR are disclosed in the art, including assays that rely on TAQMAN® probes (U.S. Pat. Nos. 5,210,015 and 5,487,972, and Lee et al., Nucleic Acids Res. 21:3761-6, 1993), molecular beacon probes (U.S. Pat. Nos. 5,925,517 and 6,103,476, and Tyagi and Kramer, Nat. Biotechnol. 14:303-8, 1996), self-probing amplicons (scorpions) (U.S. Pat. No. 6,326,145, and Whitcombe et al., Nat. Biotechnol. 17:804-7, 1999), Amplisensor (Chen et al., Appl. Environ. Microbiol. 64:4210-6, 1998), Amplifluor (U.S. Pat. No. 6,117,635, and Nazarenko et al., Nucleic Acids Res. 25:2516-21, 1997, displacement hybridization probes (Li et al., Nucleic Acids Res. 30:E5, 2002), DzyNA-PCR (Todd et al., Clin. Chem. 46:625-30, 2000), fluorescent restriction enzyme detection (Cairns et al., Biochem. Biophys. Res. Commun. 318:684-90, 2004) and adjacent hybridization probes (U.S. Pat. No. 6,174,670 and Wittwer et al., Biotechniques 22:130-1, 134-8, 1997).
In some instances, real-time PCR can result in detection of a variety of gene mutations, including for example but not limited to SNPs. In some embodiments, detection of SNPs in specific gene candidates is performed using real-time PCR, based on the use of intramolecular quenching of a fluorescent molecule by use of a tethered quenching moiety. Thus, according to exemplary embodiments, real-time PCR methods also include the use of molecular beacon technology. The molecular beacon technology utilizes hairpin-shaped molecules with an internally-quenched fluorophore whose fluorescence is restored by binding to a DNA target of interest (See, e.g., Kramer, R. et al. Nat. Biotechnol. 14:303-308, 1996). In some embodiments, increased binding of the molecular beacon probe to the accumulating PCR product is used to specifically detect SNPs present in genomic DNA.
One of the many suitable genotyping procedures is the TAQMAN® allelic discrimination assay. In some instances of this assay, an oligonucleotide probe labeled with a fluorescent reporter dye at the 5′ end of the probe and a quencher dye at the 3′ end of the probe is utilized. The proximity of the quencher to the intact probe maintains a low fluorescence for the reporter. During the PCR reaction, the 5′ nuclease activity of DNA polymerase cleaves the probe, and separates the dye and quencher. This results in an increase in fluorescence of the reporter. Accumulation of PCR product is detected directly by monitoring the increase in fluorescence of the reporter dye. The 5′ nuclease activity of DNA polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target and is amplified during PCR. The probe is designed to straddle a target SNP position and hybridize to the nucleic acid molecule only if a particular SNP allele is present.
By way of example, to amplify the Avellino corneal dystrophy associated SNP located in exon 4 of the TGFβI gene, forward and reverse PCR primer pairs (SEQ ID NOs:1 to 24 in FIG. 2 ) were constructed as described in U.S. Patent Publication No. 2012/0077200. In some embodiments, any of the forward and reverse primer pairs disclosed therein are used in the improved methods disclosed herein. In a preferred embodiment, the forward and reverse primer pair of SEQ ID NO:1 (forward) and SEQ ID NO:2 (reverse) are used in the improved methods provided herein.
In order to detect the guanine-to-adenine mutation in exon 4 of TGFβI gene, fluorescently labeled real-time PCR probe pairs for the detection of the wild type (“G”) and Avellino corneal dystrophy-associated mutant (“A”) allele having nucleotide sequences according to SEQ ID NOs: 25 to 42, as shown in FIG. 3 , were constructed as described in U.S. Patent Publication 2012/0077200. In some embodiments, any of the wild type and mutant probes are used in the improved methods disclosed herein. In a preferred embodiment, the wild type and mutant probe pair of SEQ ID NO:25 (wild type) and SEQ ID NO:26 (mutant) are used in the improved methods provided herein. To differentiate the wild type allele from the disease-associated allele, the wild type probes were labeled with VIC, and the mutant probes were labeled with FAM. The minor groove binder (MGB) was attached to the probe so as to facilitate binding to a complementary gene fragment.
b. Real-Time PCR Cycles
Real-time PCR methods include a variety of steps or cycles as part of the methods for amplification. These cycles include denaturing double-stranded nucleic acids, annealing a forward primer, a reverse primer and a detection probe to the target genomic DNA sequence and synthesizing (i.e., replicating) second-strand DNA from the annealed forward primer and the reverse primer. This three step process is referred to herein as a cycle.
In some embodiments, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 cycles are employed. In some embodiments, about 10 to about 60 cycles, about 20 to about 50 or about 30 to about 40 cycles are employed. In some embodiments, 40 cycles are employed.
In some embodiments, the denaturing double-stranded nucleic acids step occurs at a temperature of about 80° C. to 100° C., about 85° C. to about 99° C., about 90° C. to about 95° C. for about 1 second to about 5 seconds, about 2 seconds to about 5 seconds, or about 3 seconds to about 4 seconds. In some embodiments, the denaturing double-stranded nucleic acids step occurs at a temperature of 95° C. for about 3 seconds.
In some embodiments, the annealing a forward primer, a reverse primer and a detection probe to the target genomic DNA sequence step occurs at about 40° C. to about 80° C., about 50° C. to about 70° C., about 55° C. to about 65° C. for about 15 seconds to about 45 seconds, about 20 seconds to about 40 seconds, about 25 seconds to about 35 seconds. In some embodiments, the annealing a forward primer, a reverse primer and a detection probe to the target genomic DNA sequence step occurs at about 60° C. for about 30 seconds.
In some embodiments, the synthesizing (i.e., replicating) second-strand DNA from the annealed forward primer and the reverse primer occurs at about 40° C. to about 80° C., about 50° C. to about 70° C., about 55° C. to about 65° C. for about 15 seconds to about 45 seconds, about 20 seconds to about 40 seconds, about 25 seconds to about 35 seconds. In some embodiments, the annealing a forward primer, a reverse primer and a detection probe to the target genomic DNA sequence step occurs at about 60° C. for about 30 seconds.
In some embodiments, it was found that about 1 μL, about 2 μL, about 3 μL, about 4 μL or about 5 μL of a genomic DNA sample prepared according to the present methods described herein, are combined with only about 0.05 μL, about 0.10 μL about 0.15 μL, about 0.20 μL, about 0.25 μL or about 0.25 μL of a 30×, 35×, 40×, 45×, 50× or 100× real-time PCR assay mix and distilled water to form the PCR master mix. In some embodiments, the PCR master mix has a final volume of about 5 μL, about 6 μL, about 7 μL, about 8 μL, about 9 μL, about 0 μL, about 11 μL, about 12 μL, about 13 μL, about 14 μL, about 15 μL, about 16 μL, about 17 μL, about 18 μL, about 19 μL or about 20 μL or more. In some embodiments, it was found that 2 μL of a genomic DNA sample prepared as described above, are combined with only about 0.15 μL of a 40× real-time PCR assay mix and 2.85 μL of distilled water in order to form the PCR master mix.
While exemplary reactions are described herein, one of skill would understand how to modify the temperatures and times based on the probe design. Moreover, the present methods contemplate any combination of the above times and temperatures.
c. PCR Primers and Primer Design
In some embodiments, primers are tested and designed in a laboratory setting. In some embodiments, primers are designed by computer based in silico methods. Primer sequences are based on the sequence of the amplicon or target nucleic acid sequence that is to be amplified. Shorter amplicons typically replicate more efficiently and lead to more efficient amplification as compared to longer amplicons.
In designing primers, one of skill would understand the need to take into account melting temperature (T_m; the temperature at which half of the primer-target duplex is dissociated and becomes single stranded and is an indication of duplex stability; increased T_mindicates increased stability) based on GC and AT content of the primers being designed as well as secondary structure considerations (increased GC content can lead to increased secondary structure). T_M's can be calculated using a variety of methods known in the art and those of skill would readily understand such various methods for calculating T_M; such methods include for example but are not limited to those available in online tools such as the T_Mcalculators available on the World Wide Web at promega.com/techserv/tools/biomath/calc11.htm. Primer specificity is defined by its complete sequence in combination with the 3′ end sequence, which is the portion elongated by Taq polymerase. In some embodiments, the 3′ end should have at least 5 to 7 unique nucleotides not found anywhere else in the target sequence, in order to help reduce false-priming and creation of incorrect amplification products. Forward and reverse primers typically bind with similar efficiency to the target. In some instances, tools such as NCBI BLAST (located on the World Wide Web at ncbi.nlm.nih.gov) are employed to performed alignments and assist in primer design.
Those of skill in the art would be well aware of the basics regarding primer design for a target nucleic acid sequence and a variety of reference manuals and texts have extensive teachings on such methods, including for example, Molecular Cloning (three volume set, Cold Spring Harbor Laboratory Press, 2012) and Current Protocols (Genetics and Genomics; Molecular Biology; 2003-2013) and Real-Time PCR in Microbiology: From Diagnostics to Characterization (Ian M. MacKay, Calster Academic Press; 2007); PrimerAnalyser Java tool available on the World Wide Web at primerdigital.com/tools/PrimerAnalyser.html and Kalendar R, et al. (Genomics, 98(2): 137-144 (2011)), all of which are incorporated herein in their entireties for all purposes.
An additional aspect of primer design is primer complexity or linguistic sequence complexity (see, Kalendar R, et al. (Genomics, 98(2): 137-144 (2011)). Primers with greater linguistic sequence complexity (e.g., nucleotide arrangement and composition) are typically more efficient. In some embodiments, the linguistic sequence complexity calculation method is used to search for conserved regions between compared sequences for the detection of low-complexity regions including simple sequence repeats, imperfect direct or inverted repeats, polypurine and polypyrimidine triple-stranded cDNA structures, and four-stranded structures (such as G-quadruplexes). In some embodiments, linguistic complexity (LC) measurements are performed using the alphabet-capacity L-gram method (see, A. Gabrielian, A. Bolshoy, Computer & Chemistry 23:263-274 (1999) and Y. L. Orlov, V. N. Potapov, Complexity: an internet resource for analysis of DNA sequence complexity, Nucleic Acids Res. 32: W628-W633(2004)) along the whole sequence length and calculated as the sum of the observed range (xi) from 1 to L size words in the sequence divided by the sum of the expected (E) value for this sequence length. Some G-rich (and C-rich) nucleic acid sequences fold into four-stranded DNA structures that contain stacks of G-quartets (see, the World Wide Web at quadruplex.org). In some instances, these quadruplexes are formed by the intermolecular association of two or four DNA molecules, dimerization of sequences that contain two G-bases, or by the intermolecular folding of a single strand containing four blocks of guanines (see, P. S. Ho, PNAS, 91:9549-9553 (1994); I. A. Il'icheva, V. L. Florent'ev, Russian Journal of Molecular Biology 26:512-531(1992); D. Sen, W. Gilbert, Methods Enzymol. 211:191-199 (1992); P. A. Rachwal, K. R. Fox, Methods 43:291-301 (2007); S. Burge, G. N. Parkinson, P. Hazel, A. K. Todd, K. Neidle, Nucleic Acids Res. 34:5402-5415 (2006); A. Guédin, J. Gros, P. Alberti, J. Mergny, Nucleic Acids Res. 38:7858-7868 (2010); O. Stegle, L. Payet, J. L. Mergny, D. J. MacKay, J. H. Leon, Bioinformatics 25:i374-i382 (2009); in some instances, these are eliminated from primer design because of their low linguistic complexity, LC=32% for (TTAGGG)₄.
These methods include various bioinformatics tools for pattern analysis in sequences having GC skew, (G−C)/(G+C), AT skew, (A−T)/(A+T), CG−AT skew, (S−W)/(S+W), or purine-pyrimidine (R−Y)/(R+Y) skew regarding CG content and melting temperature and provide tools for determining linguistic sequence complexity profiles. For example the GC skew in a sliding window of n, where n is a positive integer, bases is calculated with a step of one base, according to the formula, (G−C)/(G+C), in which G is the total number of guanines and C is the total number of cytosines for all sequences in the windows (Y. Benita, et al., Nucleic Acids Res. 31:e99 (2003)). Positive GC-skew values indicated an overabundance of G bases, whereas negative GC-skew values represented an overabundance of C bases. Similarly, other skews are calculated in the sequence. Such methods, as well as others, are employed to determine primer complexity in some embodiments.
According to non-limiting example embodiments, real-time PCR is performed using exonuclease primers (TAQMAN® probes). In such embodiments, the primers utilize the 5′ exonuclease activity of thermostable polymerases such as Taq to cleave dual-labeled probes present in the amplification reaction (See, e.g., Wittwer, C. et al. Biotechniques 22:130-138, 1997). While complementary to the PCR product, the primer probes used in this assay are distinct from the PCR primer and are dually-labeled with both a molecule capable of fluorescence and a molecule capable of quenching fluorescence. When the probes are intact, intramolecular quenching of the fluorescent signal within the DNA probe leads to little signal. When the fluorescent molecule is liberated by the exonuclease activity of Taq during amplification, the quenching is greatly reduced leading to increased fluorescent signal. Non-limiting examples of fluorescent probes include the 6-carboxy-floruescein moiety and the like. Exemplary quenchers include Black Hole Quencher 1 moiety and the like.
A variety of PCR primers can find use with the disclosed methods. Exemplary primers include but are not limited to those described herein. Primers for use in the disclosed methods are also found in U.S. Patent Publication No. 20120077200, which is hereby incorporated by reference for all purposes. In some embodiments, the PCR primers for use in the methods of the present disclosure include but are not limited to the following listed in Table 1 and find use in the detection of the TGFβI gene. Tables 2 and 3 provide biophysical parameters for each primer, as calculated using the World Wide Web at primerdigital.com/tools/PrimerAnalyser.html.

TABLE 1

Exemplary Primers for the TGFβI gene

Primer Name	SEQ ID NO:	Primer Sequence

ACD Fw primer	SEQ ID NO: 1	5′-TCC ACC ACC ACT CAG CTG TA

ACD Re primer	SEQ ID NO: 2	5′-CCA TCT CAG GCC TCA GCT T
		(60 bp)

AV Fw primer	SEQ ID NO: 3	5′-TGC AGC CCT ACC ACT CTC AA

AV Re primer	SEQ ID NO: 4	5′-AGG CCT CGT TGC TAG G
		(150 bp)

Real Fw primer	SEQ ID NO: 5	5′-TAG TCT CTT ATT CTA ATA GA

Real Re primer	SEQ ID NO: 6	5′-GCT GCA GAC TCT GTG TTT AA
		(860 bp)

ACD Fw2 primer	SEQ ID NO: 7	5′-CCA TCC CTC CTT CTG TCT TCT G

ACD Re2 primer	SEQ ID NO: 8	5′-CGG GCC CCT CCA TCT C
		(140 bp)

ACD Fw3 primer	SEQ ID NO: 9	5′-CAG AGA AGG GAG GGT GTG GTT

ACD Re3 primer	SEQ ID NO: 10	5′-GGG CGA AGA TGG TGA AGC T
		(190 bp)

ACD Fw4 primer	SEQ ID NO: 11	5′-TCC TCG TCC TCT CCA CCT GTA

ACD Re4 primer	SEQ ID NO: 12	5′-AGC TGG CAA GGA GGC CC

ACD Fw5 primer	SEQ ID NO: 13	5′-TTT GGG CTT TCC CAC ATG C

ACD Re5 primer	SEQ ID NO: 14	5′-GGC AGA CGG AGG TCA TCT CA

ACD Fw6 primer	SEQ ID NO: 15	5′-GTA GTA CCG TGC TCT CTG

ACD Re6 primer	SEQ ID NO: 16	5′-AGT TCC CCA TAA GAA TCC CCC

ACD Fw7 primer	SEQ ID NO: 17	5′-GGC TGG ACC CCC AGA GG

ACD Re7 primer	SEQ ID NO: 18	5′-ACC CCT CGG GGA AGT AAG G

ACD Fw8 primer	SEQ ID NO: 19	5′-AAC CTT TAC GAG ACC CTG GGA

ACD Re8 primer	SEQ ID NO: 20	5′-GAC TCC CAT CCA TCA TGC CC

ACD Fw9 primer	SEQ ID NO: 21	5′-AGT CGT TGG ATC CAC CAC CA

ACD Re9 primer	SEQ ID NO: 22	5′-GAC GTC ATT TCC TAC TGT TTC AGG

ACD Fw10 primer	SEQ ID NO: 23	5′-CCC CCC AGA AAC AGC CTG

ACD Re10 primer	SEQ ID NO: 24	5′-TTC TAA GGG GTT AAG GAG AAA GCT T

GCD1 Fw primer	SEQ ID NO: 43	5′-ACA CAG TCT TTG CTC CCA CAA A

GCD1 Re primer	SEQ ID NO: 44	5′-ACT TAA GTT GGT CTT TAC CCA AGA GTC T

TABLE 2

Biophysical Parameters for Forward Primers

				GC	%	PCR
Forward Primer	Length	Tm1	Tm2	Content	Complexity	Effic.

SEQ ID NO: 1	19	55.4	57.8	57.9	70	70
SEQ ID NO: 3	20	57.1	58	55	81	66
SEQ ID NO: 5	20	40.2	45.7	25	73	38
SEQ ID NO: 7	22	55.9	60.2	54.5	62	43
SEQ ID NO: 9	21	57.5	60.2	57.1	64	40
SEQ ID NO: 11	21	57.6	60.2	57.1	66	57
SEQ ID NO: 13	19	55.4	55.7	52.6	81	80
SEQ ID NO: 15	18	50.6	55.3	55.6	75	66
SEQ ID NO: 17	17	57.8	62.2	76.5	74	60
SEQ ID NO: 19	21	56.6	58.2	52.4	82	73
SEQ ID NO: 21	20	57.4	58	55	78	46
SEQ ID NO: 23	18	56.5	59.9	66.7	69	69
Avg	19.67	54.96	57.80	56.05	72.69	59.85
Median	20	56.55	58.1	55.3	73.5	63
Std Dev	1.50	5.00	4.24	11.78	6.84	14.10

TABLE 3

Biophysical Parameters for Reverse Primers

				GC	%	PCR
Reverse Primer	Length	Tm1	Tm2	Content	Complexity	Effic.

SEQ ID NO: 2	19	55.5	57.8	57.9	72	52
SEQ ID NO: 4	16	52.1	54.5	62.5	78	78
SEQ ID NO: 6	20	52.4	53.9	45	84	41
SEQ ID NO: 8	16	55.2	59.6	75	63	53
SEQ ID NO: 10	19	56.5	57.8	57.9	78	69
SEQ ID NO: 12	17	58.5	59.8	70.6	74	66
SEQ ID NO: 14	20	57.6	60.1	60	84	74
SEQ ID NO: 16	21	54.9	58.2	52.4	71	51
SEQ ID NO: 18	19	56.6	60	63.2	78	60
SEQ ID NO: 20	20	56.5	60.1	60	65	65
SEQ ID NO: 22	24	55.5	58.7	45.8	88	67
SEQ ID NO: 24	25	55.3	57.2	40	74	40
Avg	19.69	55.61	58.13	57.33	76.54	60.69
Median	19.5	55.5	58.45	58.95	76	62.5
Std Dev	2.77	1.86	2.10	10.33	7.52	12.30

In some embodiments, the real-time PCR primers for use with the disclosed methods have a linguistic sequence complexity of at least 70%, at least 72%, at least 75%, at least 77%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97% or at least 99%.
d. Detection Probe Design and Detection Probes
A variety of detection probes can find use with the disclosed methods and are employed for genotyping and or for quantification. Detection probes commonly employed by those of skill in the art include but are not limited to hydrolysis probes (also known as TAQMAN® probes, 5′ nuclease probes or dual-labeled probes), hybridization probes, and Scorpion primers (which combine primer and detection probe in one molecule). In some embodiments, detection probe design is determined by one of skill in the art based on the desired probe target such that the probe is compatible with the PCR primers employed (e.g., primers and probes should not interfere with one another's functions in the real-time PCR assay). In some embodiments, probes are designed to have higher T_m's than the primers in order to promote efficient signal production. T_m's are calculated using any of a variety of methods known in the art and those of skill would readily understand such various methods for calculating T_m; such methods include for example those available in online tools such as the calculators available on the World Wide Web at promega.com/techserv/tools/biomath/calc11.htm. In some embodiments, the increased T_mof the detection probe provides that the detection probe has bound before the primers are elongated by the polymerase.
In some embodiments, detection probes contain various modifications. In some embodiments, detection probes include modified nucleic acid residues, such as but not limited to 2′-O-methyl ribonucleotide modifications, phosphorothioate backbone modifications, phosphorodithioate backbone modifications, phosphoramidate backbone modifications, methylphosphonate backbone modifications, 3′ terminal phosphate modifications and/or 3′ alkyl substitutions.
In some embodiments, the detection probe has increased affinity for a target sequence due to modifications. Such detection probes include detection probes with increased length, as well as detection probes containing chemical modifications. Such modifications include but are not limited to 2′-fluoro (2′-deoxy-2′-fluoro-nucleosides) modifications, LNAs (locked nucleic acids), PNAs (peptide nucleic acids), ZNAs (zip nucleic acids), morpholinos, methylphosphonates, phosphoramidates, polycationic conjugates and 2′-pyrene modifications. In some embodiments, the detector probes contains one or more modifications including 2′ fluoro modifications (aka, 2′-Deoxy-2′-fluoro-nucleosides), LNAs (locked nucleic acids), PNAs (peptide nucleic acids), ZNAs (zip nucleic acids), morpholinos, methylphosphonates, phosphoramidates, and/or polycationic conjugates.
In some embodiments, the detection probes contain detectable moieties, such as those described herein as well as any detectable moieties known to those of skill in the art. Such detectable moieties include for example but are not limited to fluorescent labels and chemiluminescent labels. Examples of such detectable moieties can also include members of FRET pairs. In some embodiments, the detection probe contains a detectable entity.
Examples of fluorescent labels include but are not limited to AMCA, DEAC (7-Diethylaminocoumarin-3-carboxylic acid); 7-Hydroxy-4-methylcoumarin-3; 7-Hydroxycoumarin-3; MCA (7-Methoxycoumarin-4-acetic acid); 7-Methoxycoumarin-3; AMF (4′-(Aminomethyl)fluorescein); 5-DTAF (5-(4,6-Dichlorotriazinyl)aminofluorescein); 6-DTAF (6-(4,6-Dichlorotriazinyl)aminofluorescein); 6-FAM (6-Carboxyfluorescein; aka FAM; including TAQMAN® FAM™); TAQMAN VIC®; 5(6)-FAM cadaverine; 5-FAM cadaverine; 5(6)-FAM ethylenediamme; 5-FAM ethylenediamme; 5-FITC (FITC Isomer I; fluorescein-5-isothiocyanate); 5-FITC cadaverin; Fluorescein-5-maleimide; 5-IAF (5-Iodoacetamidofluorescein); 6-JOE (6-Carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein); 5-CR11O (5-Carboxyrhodamine 110); 6-CR11O (6-Carboxyrhodamine 110); 5-CR6G (5-Carboxyrhodamine 6G); 6-CR6G (6-Carboxyrhodamine 6G); 5(6)-Caroxyrhodamine 6G cadaverine; 5(6)-Caroxyrhodamine 6G ethylenediamme; 5-ROX (5-Carboxy-X-rhodamine); 6-ROX (6-Carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine); 6-TAMRA (6-Carboxytetramethylrhodamine); 5-TAMRA cadaverine; 6-TAMRA cadaverine; 5-TAMRA ethylenediamme; 6-TAMRA ethylenediamme; 5-TMR C6 maleimide; 6-TMR C6 maleimide; TR C2 maleimide; TR cadaverine; 5-TRITC; G isomer (Tetramethylrhodamine-5-isothiocyanate); 6-TRITC; R isomer (Tetramethylrhodamine-6-isothiocyanate); Dansyl cadaverine (5-Dimethylaminonaphthalene-1-(N-(5-aminopentyl))sulfonamide); EDANS C2 maleimide; fluorescamine; NBD; and pyrromethene and derivatives thereof.
Examples of chemiluminescent labels include but are not limited to those labels used with Southern Blot and Western Blot protocols (see, for e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, (3rd ed.) (2001); incorporated by reference herein in its entirety). Examples include but are not limited to -(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD); acridinium esters and adamantyl-stabilized 1,2-dioxetanes, and derivatives thereof.
The labeling of probes is known in the art. The labeled probes are used to hybridize within the amplified region during amplification. The probes are modified so as to avoid them from acting as primers for amplification. The detection probe is labeled with two fluorescent dyes, one capable of quenching the fluorescence of the other dye. One dye is attached to the 5′ terminus of the probe and the other is attached to an internal site, so that quenching occurs when the probe is in a non-hybridized state.
Typically, real-time PCR probes consist of a pair of dyes (a reporter dye and an acceptor dye) that are involved in fluorescence resonance energy transfer (FRET), whereby the acceptor dye quenches the emission of the reporter dye. In general, the fluorescence-labeled probes increase the specificity of amplicon quantification.
Real-time PCR that are used in some embodiments of the disclosed methods also include the use of one or more hybridization probes (i.e., detection probes), as determined by those skilled in the art, in view of this disclosure. By way of non-limiting example, such hybridization probes include but are not limited to one or more of those provided in the described methods. Exemplary probes, such as the HEX channel and/or FAM channel probes, are understood by one skilled in the art.
According to example embodiments, detection probes and primers are conveniently selected e.g., using an in silico analysis using primer design software and cross-referencing against the available nucleotide database of genes and genomes deposited at the National Center for Biotechnology Information (NCBI). Some additional guidelines may be used for selection of primers and/or probes in some embodiments. For example, in some embodiments, the primers and probes are selected such that they are close together, but not overlapping. In some embodiments, the primers may have the same (or close T_M) (e.g., between about 58° C. and about 60° C.). In some embodiments, the T_Mof the probe is approximately 10° C. higher than that selected for the T_Mof the primers. In some embodiments, the length of the probes and primers is selected to be between about 17 and 39 base pairs, etc. These and other guidelines are used in some instances by those skilled in the art in selecting appropriate primers and/or probes.
Probes for use in the methods of the present invention include but are not limited to the following exemplary probes listed in Table 4.

TABLE 4

Exemplary Probes for the TGFβI gene

Probe Name	SEQ ID NO:	Probe Sequence

Normal probe 1	SEQ ID NO: 25	VIC-CAC GGA CCG CAC GGA-NFQ
		(15 bp)

Mutant probe 1	SEQ ID NO: 26	FAM-CAC GGA CCA CAC GGA-NFQ

Normal probe 2	SEQ ID NO: 27	VIC-ACA CGG ACC GCA CG-NFQ

Mutant probe 2	SEQ ID NO: 28	FAM-ACA CGG ACC ACA CG-NFQ
		(14 bp)

Normal probe 3	SEQ ID NO: 29	VIC-TAC ACG GAC CGC A-NFQ

Mutant probe 3	SEQ ID NO: 30	FAM-TAC ACG GAC CAC A-NFQ
		(13 bp)

Normal probe 4	SEQ ID NO: 31	VIC-CTG TAC ACG GAC CGC ACG-NFQ

Mutant probe 4	SEQ ID NO: 32	FAM-CTG TAC ACG GAC CAC ACG-NFQ
		(18 bp)

Normal probe 5	SEQ ID NO: 33	VIC-CTG TAC ACG GAC CGC ACG GAG-NFQ

Mutant probe 5	SEQ ID NO: 34	FAM-CTG TAC ACG GAC CAC ACG GAG-NFQ
		(21 bp)

Normal probe 6	SEQ ID NO: 35	VIC-GCT GTA CAC GGA CCG CAC GGA GAA-
		NFQ

Mutant probe 6	SEQ ID NO: 36	FAM-GCT GTA CAC GGA CCA CAC GGA GM-
		NFQ

Normal probe 7	SEQ ID NO: 37	VIC-ACC GCA CGG AGA AGC-NFQ

Mutant probe 7	SEQ ID NO: 38	FAM-ACC ACA CGG AGA AGC-NFQ

Normal probe 8	SEQ ID NO: 39	VIC-ACC GCA CGG AGA AGC TGA GGC-NFQ

Mutant probe 8	SEQ ID NO: 40	FAM-ACC ACA CGG AGA AGC TGA GGC-NFQ

Normal probe 9	SEQ ID NO: 41	VIC-ACC GCA CGG AGA AGC TGA GGC CTG-
		NFQ

Mutant probe 9	SEQ ID NO: 42	FAM-ACC ACA CGG AGA AGC TGA GGC CTG-
		NFQ

Normal Probe 10	SEQ ID NO: 45	VIC-CAC CAA GAG AAC GGA-NFQ

Mutant Probe 10	SEQ ID NO: 46	FAM-CAC CAA GAG AAT GG-NFQ

Normal Probe 10a	SEQ ID NO: 47	VIC-CAC CAA GAG AAC GGA G-MGB NFQ

#TGFBI R124C	SEQ ID NO: 48	FAM-CAC GGA CTG CAC GGA-MGB NFQ

#TGFBI R124L	SEQ ID NO: 49	FAM-CAC GGA CCT CAC GGA-MGB NFQ

#TGFBI R555Q-1	SEQ ID NO: 50	FAM-AC CAA GAG AACA GA G--MGB NFQ

VII. Diagnostic Tests
In some embodiments, diagnostic testing is employed to determine one or more genetic conditions by detection of any of a variety of mutations. In some embodiments, diagnostic testing is used to confirm a diagnosis when a particular condition is suspected based on for example physical manifestations, signs and/or symptoms as well as family history information. In some embodiments, the results of a diagnostic test assist those of skill in the medical arts in determining an appropriate treatment regimen for a given patient and allow for more personalized and more effective treatment regimens. In some embodiments, a treatment regimen include any of a variety of pharmaceutical treatments, surgical treatments, lifestyles changes or a combination thereof as determined by one of skill in the art.
The nucleic acids obtained by the disclosed methods are useful in a variety of diagnostic tests, including tests for detecting mutations such as deletions, insertions, transversions and transitions. In some embodiments, such diagnostics are useful for identifying unaffected individuals who carry one copy of a gene for a disease that requires two copies for the disease to be expressed, identifying unaffected individuals who carry one copy of a gene for a disease in which the information could find use in developing a treatment regimen, preimplantation genetic diagnosis, prenatal diagnostic testing, newborn screening, genealogical DNA test (for genetic genealogy purposes), presymptomatic testing for predicting adult-onset disorders such as Huntington's disease, presymptomatic testing for estimating the risk of developing adult-onset cancers and Alzheimer's disease, confirmational diagnosis of a symptomatic individual, and/or forensic/identity testing. In some embodiments, the present methods find use in the detection of corneal dystrophy. In some embodiments, corneal dystrophy is detected for example through detection of Avellino corneal dystrophy-related SNPs, such as those that result in R124 mutations in the TGFβI gene (including for example but not limited to an R124H mutation caused by a G to A transition at nucleotide 418 of TGFβI gene also referred to as a C(G/A)C SNP). In some embodiments, corneal dystrophy is detected for example through detection of granular corneal dystrophy-related SNPs, such as those that result in R555 mutations in the TGFβI gene (including for example but not limited to an R555W mutation caused by a C to T transition at nucleotide 1663 of TGFβI gene also referred to as a (C/T)GG SNP). In some embodiments, corneal dystrophy is detected for example through detection of lattice dystrophy-related SNPs, such as those that result in R124 and/or 626 mutations in the TGFβI gene (including for example but not limited to an R124C mutation caused by a C to T transition at nucleotide 417 of TGFβI gene also referred to as a (C/T)GC SNP or a H626P mutation caused by an A to C transition at nucleotide 1924 of TGFβI gene. In some embodiments, corneal dystrophy is detected for example through detection of Reis-Buckler corneal dystrophy-related SNPs, such as those that result in R124 mutations in the TGFβI gene (including for example but not limited to an R124L mutation caused by a G to T transition at nucleotide 418 of TGFβI gene also referred to as a C(G/T)C SNP). In some embodiments, corneal dystrophy is detected for example through detection of Thiel-Behnke corneal dystrophy-related SNPs, such as those that result in R555 mutations in the TGFβI gene (including for example but not limited to an R555Q mutation caused by a G to A transition at nucleotide 1664 of TGFβI gene also referred to as a C(G/A)G SNP).
In some embodiments, newborn screening includes any genetic screening employed just after birth in order to identify genetic disorders. In some embodiments, newborn screening finds use in the identification of genetic disorders so that a treatment regimen is determined early in life. Such tests include but are not limited to testing infants for phenylketonuria and congenital hypothyroidism.
In some embodiments, carrier testing is employed to identify people who carry a single copy of a gene mutation. In some cases, when present in two copies, the mutation can cause a genetic disorder. In some cases, one copy is sufficient to cause a genetic disorder. In some cases, the presence of two copies is contra-indicated for a particular treatment regimen, such as the presence of the Avellino mutation and pre-screening prior to performing surgical procedures in order to ensure the appropriate treatment regiment is pursued for a given patient. In some embodiments, such information is also useful for individual contemplating procreation and assists individuals with making informed decisions as well as assisting those skilled in the medical arts in providing important advice to individual patients as well as patients' relatives.
In some embodiments, predictive and/or presymptomatic types of testing are used to detect gene mutations associated with a variety of disorders. In some cases, these tests are helpful to people who have a family member with a genetic disorder, but who may exhibit no features of the disorder at the time of testing. In some embodiments, predictive testing identifies mutations that increase a person's chances of developing disorders with a genetic basis, including for example but not limited to certain types of cancer. In some embodiments, presymptomatic testing is useful in determining whether a person will develop a genetic disorder, before any physical signs or symptoms appear. The results of predictive and presymptomatic testing provides information about a person's risk of developing a specific disorder and help with making decisions about an appropriate medical treatment regimen for a patient as well as for a patient's relatives. Predictive testing is also employed, in some embodiments, to detect mutations which are contra-indicated with certain treatment regimens, such as the presence of the Avellino mutation being contra-indicated with performing LASIK surgery and/or other refractive procedures, such as but not limited to Phototherapeutic keratectomy (PTK) and/or Photorefractive keratectomy (PRK). For example, patients exhibiting the Avellino mutation should not undergo LASIK surgery or other refractive procedures.
In some embodiments, diagnostic testing also includes pharmacogenomics which includes genetic testing that determines the influence of genetic variation on drug response. Information from such pharmacogenomic analyses finds use in determining and developing an appropriate treatment regimen. Those of skill in the medical arts employ information regarding the presence and/or absence of a genetic variation in designing appropriate treatment regimen.
In some embodiments, diseases whose genetic profiles are determined using the methods of the present disclosure include but are not limited to ophthalmic disorders, cancer, diabetes mellitus, hypertension, schizophrenia, and most common congenital malformations, such as cleft lip, cleft palate, neural tube defects, Achondroplasia, Alpha-1 Antitrypsin Deficiency, Antiphospholipid Syndrome, Autism, Autosomal Dominant Polycystic Kidney Disease, Charcot-Marie-Tooth, Colon cancer, Cri du chat, Crohn's Disease, Cystic fibrosis, Dercum Disease, Down Syndrome, Duane Syndrome, Duchenne Muscular Dystrophy, Factor V Leiden Thrombophilia, Familial Hypercholesterolemia, Familial Mediterranean Fever, Fragile X Syndrome, Gaucher Disease, Hemochromatosis, Hemophilia, Holoprosencephaly, Huntington's disease, Klinefelter syndrome, Marfan syndrome, Myotonic Dystrophy, Neurofibromatosis, Noonan Syndrome, Osteogenesis imperfecta, Parkinson's disease, Phenylketonuria, Poland Anomaly, Porphyria, Progeria, Retinitis Pigmentosa, Severe Combined Immunodeficiency (SCID), Sickle cell disease, Spinal Muscular Atrophy, Tay-Sachs, Thalassemia, Trimethylaminuria, Turner Syndrome, Velocardiofacial Syndrome, WAGR Syndrome, Wilson Disease, as well as any other disease with a genetic component. Ophthalmic disorders and/or disorders that include an ophthalmic component include but are not limited to chalazion, stye, trichiasis, entropion, ectropion, lagophthalmos, bleharitis, dacryocystitis, orbital cellulitis, ptergium, pterygiumcorneal dystrophy, conjuctivitis, ophtalmia neonatorum, bacterial corneal ulcer, fungal corneal ulcer, glaucoma, Fuchs Dystrophy, keratoconus, Advanced Macular Degeneration, Retinitis pigmentosa, cataracts, retinal disorecers, macular degeneration, diabetic eye problems (for example, diabetic retinopathy), blepharophimosis-ptosis-epicanthus-inversus syndrome (BPES), oculocutaneous albinism, Marfan syndrome, Stickler syndrome, and CHARGE (coloboma, heart anomalies, atresia of the choanae, retardation of growth and development, genital/urinary anomalies, ear abnormalities or deafness) syndrome. Corneal dystrophies include but are not limited to Avellino corneal dystrophy, granular corneal dystrophy, lattice type I corneal dystrophy, Fuchs Dystrophy, Thiel-Behnke and Reis-bucklers corneal dystrophy. Cancers include but are not limited to carcinoma, sarcoma, blastoma, lymphoma, leukemia germ cell tumors, and cancers of unknown origin. In some embodiments, the cancer include but is not limited to head and neck, skin, colon, oral, glioblastoma, breast, laryngeal, esophageal, endothelial, endometrial, ovarian, lung, urogenital, rectal, prostate, kidney, melanoma, renal, pancreatic, gastrointestinal, blood, liver, uterine and brain as well as viral induced cancers such as papilloma virus-induced cancer.
In some embodiments, the present methods find use in development of personalized medicine treatment regimens by providing the genomic DNA which is used in determining the genetic profile for an individual. In some embodiments, such genetic profile information is employed by those skilled in the art in order determine and/or develop a treatment regimen. In some embodiments, the presence and/or absence of various genetic variations and mutations identified in nucleic acids isolated by the described methods are used by those of skill in the art as part of a personalized medicine treatment regimen or plan. For example, in some embodiments, information obtained using the disclosed methods is compared to databases or other established information in order to determine a diagnosis for a specified disease and or determine a treatment regimen. In some cases, the information regarding the presence or absence of a genetic mutation in a particular patient is compared to a database or other standard source of information in order to make a determination regarding a proposed treatment regimen. In some cases, the presence of a genetic mutation indicates pursuing a particular treatment regimen. In some cases the absence of a genetic mutation indicates not pursuing a particular treatment regimen.
In some embodiments, information regarding the presence and/or absence of a particular genetic mutation is used to determine the treatment efficacy of treatment with the therapeutic entity, as well as to tailor treatment regimens for treatment with therapeutic entity. In some embodiments, information regarding the presence and/or absence of a genetic mutation is employed to determine whether to pursue a treatment regimen. In some embodiments, information regarding the presence and/or absence of a genetic mutation is employed to determine whether to continue a treatment regimen. In some embodiments, the presence and/or absence of a genetic mutation is employed to determine whether to discontinue a treatment regimen. In other embodiments, the presence and/or absence of a genetic mutation is employed to determine whether to modify a treatment regimen. In some embodiments the presence and/or absence of a genetic mutation is used to determine whether to increase or decrease the dosage of a treatment that is being administered as part of a treatment regimen. In other embodiments, the presence and/or absence of a genetic mutation is used to determine whether to change the dosing frequency of a treatment administered as part of a treatment regimen. In some embodiments, the presence and/or absence of a genetic mutation is used to determine whether to change the number of dosages per day, per week, times per day of a treatment. In some embodiments the presence and/or absence of a genetic mutation is used to determine whether to change the dosage amount of a treatment. In some embodiments, the presence and/or absence of a genetic mutation is determined prior to initiating a treatment regiment and/or after a treatment regimen has begun. In some embodiments, the presence and/or absence of a genetic mutation is determined and compared to predetermined standard information regarding the presence or absence of a genetic mutation.
In some embodiments, a composite of the presence and/or absence of more than one genetic mutation is generated using the disclosed methods and such composite includes any collection of information regarding the presence and/or absence of more than one genetic mutation. In some embodiments, the presence or absence of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more or 40 or more genetic mutations is examined and used for generation of a composite. Exemplary information in some embodiments includes nucleic acid or protein information, or a combination of information regarding both nucleic acid and/or protein genetic mutations. Generally, the composite includes information regarding the presence and/or absence of a genetic mutation. In some embodiments, these composites are used for comparison with predetermined standard information in order to pursue, maintain or discontinue a treatment regimen.
In some embodiments, corneal dystrophy is detected for example through detection of 2, 3, 4 or 5 SNPs selected from but not limited to Avellino corneal dystrophy-related SNPs, granular corneal dystrophy-related SNPs, lattice dystrophy-related SNPs, Reis-Buckler corneal dystrophy-related SNPs, and/or Thiel-Behnke corneal dystrophy-related SNPs. In some embodiments, corneal dystrophy is detected for example through detection of an Avellino corneal dystrophy-related SNP, in combination with a granular corneal dystrophy-related SNP, a lattice dystrophy-related SNP, a Reis-Buckler corneal dystrophy-related SNP, and/or a Thiel-Behnke corneal dystrophy-related SNP. In some embodiments, corneal dystrophy is detected for example through detection of a granular corneal dystrophy-related SNP in combination with an Avellino corneal dystrophy-related SNP, a lattice dystrophy-related SNPs, a Reis-Buckler corneal dystrophy-related SNPs, and/or a Thiel-Behnke corneal dystrophy-related SNP. In some embodiments, corneal dystrophy is detected for example through detection of a lattice dystrophy-related SNP in combination with an Avellino corneal dystrophy-related SNP, a granular corneal dystrophy-related SNP, a Reis-Buckler corneal dystrophy-related SNP, and/or a Thiel-Behnke corneal dystrophy-related SNP. In some embodiments, corneal dystrophy is detected for example through detection of a Reis-Buckler corneal dystrophy-related SNP in combination with an Avellino corneal dystrophy-related SNP, a granular corneal dystrophy-related SNP, a lattice dystrophy-related SNP, and/or a Thiel-Behnke corneal dystrophy-related SNP. In some embodiments, corneal dystrophy is detected for example through detection of a Thiel-Behnke corneal dystrophy-related SNP in combination with an Avellino corneal dystrophy-related SNP, a granular corneal dystrophy-related SNP, a lattice dystrophy-related SNP and/or a Reis-Buckler corneal dystrophy-related SNP.
In some embodiments, corneal dystrophy is detected for example through detection of 2, 3, 4, 5 and/or 6 SNPs selected from Avellino corneal dystrophy-related SNPs. In some embodiments, the SNPs include SNPs that result in mutations at positions 124, 555 and/or 626 of the polypeptide encoded by the human TGFβI gene. These mutations include those that result in R124 mutations in the TGFβI gene (including for example but not limited to an R124H mutation caused by a G to A transition at nucleotide 418 of TGFβI gene also referred to as a C(G/A)C SNP), granular corneal dystrophy-related SNPs, such as those that result in R555 mutations in the TGFβI gene (including for example but not limited to an R555W mutation caused by a C to T transition at nucleotide 1663 of the TGFβI gene also referred to as a (C/T)GG SNP), lattice dystrophy-related SNPs, such as those that result in R124 mutations in the TGFβI gene (including for example but not limited to an R124C mutation caused by a C to T transition at nucleotide 417 of TGFβI gene also referred to as a (C/T)GC SNP), Reis-Buckler corneal dystrophy-related SNPs, such as those that result in R124 mutations in the TGFβI gene (including for example but not limited to an R124L mutation caused by a G to T transition at nucleotide 418 of TGFβI gene also referred to as a C(G/T)C SNP) and/or Thiel-Behnke corneal dystrophy-related SNPs, such as those that result in R555 mutations in the TGFβI gene (including for example but not limited to an R555Q mutation caused by a G to A transition at nucleotide 1664 of TGFβI gene also referred to as a C(G/A)G SNP). In some embodiments, corneal dystrophy is detected for example through detection of Avellino corneal dystrophy-related SNPs, such as those that result in R124 mutations in the TGFβI gene (including for example but not limited to an R124H mutation caused by a G to A transition at nucleotide 418 of TGFβI gene also referred to as a C(G/A)C SNP) in combination with a granular corneal dystrophy-related SNPs, such as those that result in R555 mutations in the TGFβI gene (including for example but not limited to an R555W mutation caused by a C to T transition at nucleotide 1663 of TGFβI gene also referred to as a (C/T)GG SNP). In some embodiments, corneal dystrophy is detected for example through detection of Avellino corneal dystrophy-related SNPs, such as those that result in H626P mutations in the TGFβI gene (including for example but not limited to an H626P mutation caused by an A to C transition at nucleotide 1924 of TGFβI gene).
In some embodiments, corneal dystrophy is detected for example through detection of 2, 3, 4, 5 and/or 6 of R124C, R124H, R124L, R555W, R555Q and/or H626P. In some embodiments, corneal dystrophy is detected for example through detection of 2, 3, 4 and/or 5 of R124C, R124H, R124L, R555W, and/or R555Q. In some embodiments, corneal dystrophy is detected for example through detection of R124C in combination with one or more of R124H, R124L, R555W and/or R555Q. In some embodiments, corneal dystrophy is detected for example through detection of R124H in combination with one or more of R124C, R124L, R555W and/or R555Q. In some embodiments, corneal dystrophy is detected for example through detection of R555W in combination with one or more of R124C, R124H, R124L and/or R555Q. In some embodiments, corneal dystrophy is detected for example through detection of R555Q in combination with one or more of R124C, R124H, R124L and/or R555W. In some embodiments, R124H is detected in combination with R555W. In some embodiments, corneal dystrophy is detected for example through detection of H262P in combination with one or more of R124C, R124H, R124L, R555W and/or R555Q. In some embodiments, R124C, R124H, R124L, R555W and/or R555Q are all detected. In some embodiments, R124C, R124H, R124L, R555W, R555Q and/or H626P are all detected.
VIII. Diagnostic Kits
In some embodiments, any or all of the reagents described above are packaged into a diagnostic kit. Such kits include any and/or all of the primers, probes, buffers and/or other reagents described herein in any combination. In some embodiments, the kit includes reagents for detection of 2, 3, 4, 5 and/or 6 of R124C, R124H, R124L, R555W, R555Q and/or H626P. In some embodiments, the kit includes reagents for detection of 2, 3, 4, and/or 5 of R124C, R124H, R124L, R555W and R555Q. In some embodiments, the kit includes reagents for detection of R124C, R124H, and R124L. In some embodiments, the kit includes reagents for detection of R555W and R555Q. In some embodiments, the kit includes reagents for detection of R124C. In some embodiments, the kit includes reagents for detection of R124H. In some embodiments, the kit includes reagents for detection of R124L. In some embodiments, the kit includes reagents for detection of R555W. In some embodiments, the kit includes reagents for detection of R555Q.
In some embodiments, the kit includes primers and probes for detection of 2, 3, 4, 5 and/or 6 of R124C, R124H, R124L, R555W, R555Q and/or H626P. In some embodiments, the kit includes primers and probes for detection of 2, 3, 4, and/or 5 of R124C, R124H, R124L, R555W and R555Q. In some embodiments, the kit includes primers and probes for detection of R124C, R124H, and R124L. In some embodiments, the kit includes primers and probes for detection of R555W and R555Q. In some embodiments, the kit includes primers and probes for detection of R124C. In some embodiments, the kit includes primers and probes for detection of R124H. In some embodiments, the kit includes primers and probes for detection of R124L. In some embodiments, the kit includes primers and probes for detection of R555W. In some embodiments, the kit includes primers and probes for detection of R555Q. In some embodiments, the kit includes primers and probes for detection of H626P.
In some embodiments, the reagents in the kit are included as lyophilized powders. In some embodiments, the reagents in the kit are included as lyophilized powders with instructions for reconstitution. In some embodiments, the reagents in the kit are included as liquids. In some embodiments, the reagents are included in plastic and/or glass vials or other appropriate containers. In some embodiments the primers and probes are all contained in individual containers in the kit. In some embodiments, the primers are packaged together in one container, and the probes are packaged together in another container. In some embodiments, the primers and probes are packaged together in a single container.
In some embodiments, the kit further includes control gDNA and/or DNA samples. In some embodiments the control DNA sample included is TGFBI R124 normal. In some embodiments the control DNA sample included corresponds to the mutation being detected, including R124C, R124H, R124L, R555W, R555Q and/or H626P. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and a mutant DNA sample corresponding to R124C, R124H, R124L, R555W, R555Q and/or H626P are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and a mutant DNA sample corresponding to R124C, R124H, R124L, R555W and/or R555Q are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and a mutant DNA sample corresponding to R124C, R124H and/or R124L are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and a mutant DNA sample corresponding to R555W and/or R555Q are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and a mutant DNA sample corresponding to R124C are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal DNA and a mutant DNA sample corresponding to R124H are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and a mutant DNA sample corresponding to R124L are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal DNA and a mutant DNA sample corresponding to R555W are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and mutant DNA sample corresponding to R555Q are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and mutant DNA sample corresponding to H626P are included. In some embodiments, the concentration of the control DNA sample is 5 ng/μL, 10 ng/μL, 20 ng/μL, 30 ng/μL, 40 ng/μL, 50 ng/μL, 60 ng/μL, 70 ng/μL, 80 ng/μL, 90 ng/μL, 100 ng/μL, 110 ng/μL, 120 ng/μL, 130 ng/μL, 140 ng/μL, 150 ng/μL, 160 ng/μL, 170 ng/μL, 180 ng/μL, 190 ng/μL or 200 ng/μL. In some embodiments, the concentration of the control DNA sample is 50 ng/μL, 100 ng/μL, 150 ng/μL or 200 ng/μL. In some embodiments, the concentration of the control DNA sample is 100 ng/μL. In some embodiments, the control DNA samples have the same concentration. In some embodiments, the control DNA samples have different concentrations.
In some embodiments, the kit can further include buffers, for example, GTXpress TAQMAN® reagent mixture, or any equivalent buffer. In some embodiments, the buffer includes any buffer described herein.
In some embodiments, the kit can further include reagents for use in cloning, such as vectors (including, e.g., M13 vector).
In some embodiments, the kit further includes reagents for use in purification of DNA.
In some embodiments, the kit further includes instructions for using the kit for the detection of corneal dystrophy in a subject. In some embodiments, these instructions include various aspects of the protocols described herein.

EXAMPLES

Example 1: DNA Extraction (DNA Extract All Reagents, ThermoFisher)

DNA was extracted from oral epithelium or hair root or whole blood as described below and in line with the disclosures provided herein.
For DNA extraction from oral epithelium or hair root, the sample was first pre-treated in 1×PBS 300 μL. Next, 30 μL Lysis solution was added to tube and the mixture vortexed. The mixture was then incubated at 95° C. for 3 min. Next, 30 μL of DNA Stabilizing Solution (from Life Technologies/Thermo Scientific, USA) was added and the mixture vortexed. The mixture was then centrifuged at 13,000 RPM for 1 min.
For DNA extraction from whole blood, the sample was first pre-treated in 1×PBS 300 μL starting with 3 μL of whole blood. Next, 30 μL of lysis solution was added to tube and the mixture vortexed. The mixture was then incubated at 95° C. for 3 min. Next, 30 μL of DNA Stabilizing Solution (from Life Technologies/Thermo Scientific, USA) was added and the mixture vortexed. The mixture was then centrifuged at 13,000 RPM for 1 min.
After the above procedures were completed, the DNA concentration was read using a commercially available Tecan® Infinite® 200 PRO NanoQuant. For quantitation, 100 μL of eluents were pipetted into a clear 96 well plate. Next, 100 μL of prepared blank solution was added to well H12. Concentrations were then read using the manufacturer's instructions provided with the NanoQuant.
Reaction mixtures were prepared using the probes and primers described below in Tables 5 and 6.

TABLE 5

Probe Sequences

	PROBE	SEQUENCE

	TGFBI R124Normal	VIC-CAC GGA CCG
	(SEQ ID NO: 25)	CAC GGA-MGB NFQ

	TGFBI R124H	FAM-CAC GGA CCA
	(SEQ ID NO: 26)	CAC GGA-MGB NFQ

	TGFBI R124C	FAM-CAC GGA CTG
	(SEQ ID NO: 48)	CAC GGA-MGB NFQ

	TGFBI R124L	FAM-CAC GGA CCT
	(SEQ ID NO: 49)	CAC GGA-MGB NFQ

	TGFBI R555Normal	VIC-CAC CAA GAG
	(SEQ ID NO: 45)	AAC GGA-MGB NFQ

	TGFBI R555W	FAM-CAC CAA GAG
	(SEQ ID NO: 46)	AAT GG-MGB NFQ

	TGFBI R555Q	FAM-AC CAA GAG
	(SEQ ID NO: 50)	AAC AGA G-MGB NFQ

TABLE 6

Primer Sequences

	PRIMER	SEQUENCE

	TGFBI
124 F	TCC ACC ACC ACT CAG
	(SEQ ID NO: 1)	CTG TA

	TGFBI
124 R	CCA TCT CAG GCC TCA
	(SEQ ID NO: 2)	GCT T

	TGFBI
555 F	ACA CAG TCT TTG CTC
	(SEQ ID NO: 43)	CCA CAA A

	TGFBI 555 R	ACT TAA GTT GGT CTT
	(SEQ ID NO: 44)	TAC CCA AGA GTC T

The components and ratios used in an exemplary reaction mixture are shown below in Table 7.

TABLE 7

Reaction Mixture

All reagent extract (whole blood)		3.00 μl
GTXpress TaqMan ®		10.00 μl
Primer (124R) (reverse)	10 pMol	0.50 μl
Primer (124F) (forward)	10 pMol	0.50 μl
Primer (555R) (reverse)	10 pMol	0.50 μl
Primer (555F) (forward)	10 pMol	0.50 μl
Probe (NL/VIC)	10 pMol	0.20 μl
Probe (R124H/FAM)	10 pMol	0.30 μl
Probe (R124C/FAM)	10 pMol	0.20 μl
Probe (R124L/FAM)	10 pMol	0.50 μl
Probe (R555N/VIC)	10 pMol	0.20 μl
Probe (R555Q-1/FAM)	10 pMol	0.20 μl
Probe (R555W/FAM)	10 pMol	0.20 μl
DW		3.20 μl

Exemplary PCR cycling conditions are shown below in Table 8.

TABLE 8

PCR Cycling

Pre-read

Hold stage

Cycling Stage

40 Cycle

Post-read

60° C.	95° C.	95° C.	64° C.	60° C.

1 min	20 sec	3 sec	30 sec	1 min

The above protocols were used to generate the Real-Time PCR data provided in FIGS. 4-8 .

REFERENCES

All headings and section designations are used for clarity and reference purposes only and are not to be considered limiting in any way. For example, those of skill in the art will appreciate the usefulness of combining various aspects from different headings as appropriate according to the spirit and scope of the invention described herein.
All references cited herein are hereby incorporated by reference herein in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled.

1 gcttgcccgt cggtcgctag ctcgctcggt gcgcgtcgtc ccgctccatg gcgctcttcg

61 tgcggctgct ggctctcgcc ctggctctgg ccctgggccc cgccgcgacc ctggcgggtc

121 ccgccaagtc gccctaccag ctggtgctgc agcacagcag gctccggggc cgccagcacg

181 ggtaagccga gccgcctggc caggggctgc ggaaggtcag gtagtcgggg ctcggagcgc

241 aagccgctgg gggcattgaa ctgggctggg ggcgcagggg acaaagcccg aactaaaaac

301 cttgcagcat ggagcgctcg gacaccagcc ctgcacgcgg tggaaggaga gagggaggga

361 ggtggaggac catggaggga aagcgggagg ccgccgcttt gtagaaggga gtggggaagt

421 ggaccagaga ctttcgacgc aggccaagag cctgagacgg acagcgcttt cagcttctcc

481 tcccagccac tgcagaaagg gggaaatggc aactctttgg ccataatcac cgtgggaggg

541 tcccaagggc aaagcccacc cagcagtaca cctattccaa cccagccagg cccccggcca

601 gcgactccag acaagaacct gggccacaca cggtggcagc atctaaggtg ccccaggctc

661 ctgtgctcct ggccaggccc tgcactcaga cactgctggc acccgacact gctctctggg

721 tacagcaagg gcaatgtggc acttcttgtc ctgcccgatg aagagcagga gaatgcactg

781 ggccctcaca cacactgttc aaatggggaa actgagtcct gagtggttcc actttcccac

841 agtcctgaag tgtgcactgg agccaggatt ggagtctgtc ttaaagtaat agctgggttt

901 gtaaatgtag gacactatca ttgcaggaat tcctttgaga ccctgaagat gtgttggctt

961 taggagacaa actcaagcag aaggtctggt ctgatagtgg ccctaatact gacccaggca

1021 gaggcaggca acatttctac ctcaaaaacc aggccatacc tgcgtcacaa atacccaggc

1081 tttgctgcag cttccagcct acctggttgc accaacttct ttttcataac taggtaaaac

1141 tatatatgag tagaatcttg tagtgactcc tcagaggaag cctaaatacc atcggggtct

1201 ggcgttcaca cccacaagca atgcccaaac ctccaagaga ctgggcagat ctgtgctcaa

1261 atcaaaactc attgttgggg gtgatagagt tgacttcaca ggccctgaaa gtcttggctc

1321 cttgcactag gagtgctctg ggtacgggta caggctgccc cttgtagggc atagttgctc

1381 ttgtttcctc tacttgtggc tttatggtct aggcctttca ggagtttggg gctctggcgg

1441 agagggcctg ctgggagcac atctggccac cctgcagagt gaaatcaaac caggcctggc

1501 tccaacctca acaccctcct ggaaagagga gaatactggg gatatcctgg ggtctttctg

1561 gaagtgggag aatcagcttt gacttgggca gtgtgcagaa tagagtgagg ggggatgtca

1621 gaaagatgag agggatatga ggcctcaaca tcaaaatgca agcacctggc atttttatta

1681 tctctgccca cctctccgtt ggtctctctg cctttcctgc caatgaattg tgttatgttt

1741 gggtgcctca atttgcctag gagggttcta tttcttctgt atcttcgcca ctaagtcagg

1801 agaagatcct tatagcatgc cctgcaacag tgtcacctgt aagggcatct ctctgcacag

1861 ccacagtgaa ggatcctcaa aggtattgag ggctttccat caagagccat ctttacagca

1921 aacctctttc ccttcagagc ccagaagagt gctgaccagc tggaaaacag ggtttttttc

1981 ttaaatgcag atgctcttga ttatgagttc cagatattag atcaacttcc ccaccatacc

2041 cctgcaggca aagcctctta attagcttcc tgcagcacag ctggaaaggc ctattgtaat

2101 ctgtgatggg cagagtaatc taagaagtca caggagcacc cctgtcccag tagaatctgg

2161 atgcgcaggc acatgaacca tggcaaaatg gttgcaggca cagttgtatt tactctgatc

2221 taactgtccc tcttaatgcc acagggctgc ctggcctggc acacagggct gtggcgcctt

2281 gtgcaaatgg ataacgttgt tctagctcca gcctttcatt caaagtgaaa actgttagaa

2341 agggaaggaa aactttgcta ttttaaggaa ttgtagcgtg ctgcctgata tgaaggaaga

2401 aataacagct gtgccttgct tgtgcgcagc actcgattgc cgcttttgct ttcgacctca

2461 ccacaacaca gtgagatcta ctgttcatgt tcccatttta caggaggtga aactgcagct

2521 tagtgaggta gagagtgact tagttcagac acagaatgct gttgggagag taataactat

2581 gatatggtct cttgactccc agctatatct gtgttgctat agggaagggg aaaaataata

2641 ctgaaagaga agtaaaaata caatcacact tccaaacatc aaccaccaaa aactgaactg

2701 aatttcctga agcacttggt tttcaaatct aagctgaaca tcaatgctgt tattcttgag

2761 gcccagaagc aacttgctca tttcaattaa gcttcagcat gaacttccta tgtacacagc

2821 ccacccacac tccccgatgt gagaaggaga gggtcacagc cgcccccagc ctctgctgct

2881 gccacaagga cagcagcagt ggaaacattc agcaaaggaa tcttggagcc acatccacaa

2941 gagactcact gaagattcgc caaacgccta cggaaagtgg cagggaattc attgacagta

3001 attgtttcct gcttgatcag attgaagagc ttctgggatt ctgtaacaat aaataggacc

3061 gggggctgga gtatggccag caaggactct tcaggggtta ttcagggact gtctaacctg

3121 tgaatcctag gcagcaaaca gaaaccaggt attcagaaat ctggaggatt tggtcaggcc

3181 cagctaggac tagggaggca tgggcctctg ctggctgtgg tcccttctcc agccttcact

3241 tctcttgtcc ctagatcctt acatggattc attaatgctc attgtccctc ctgggcccac

3301 tcactttcac ctgttgaaca aaaaactggc caagaggtga cagtcatatc accgcagaag

3361 agacagggca gagaaatgaa ggggcagaat ggactcccac ccaaaagcct gactctgaat

3421 atttgagaat tgttcaagtt cctgcagagg aatcatgatg gggacagtag gtgtagtttt

3481 tactgcaata ttggtgtctt cttaacaaat acgctgcaca tcaagtgatg tctgtggatg

3541 gcattcttaa agtaacaggg aaattgatgt taaagaaata cttcatcctt tgggtgatac

3601 ctgaagttct ctgagcttgg aggtcttgtg aaagccctca gtattgtttg ttttatttgc

3661 tttcctctga cttgtgattc agtcagatgc atgcctgcct ctggctcagg aagatcaacc

3721 ctctcctgac tgaccacgcc tctcctgact gaccacgtag cacagcagct tcctttccct

3781 aggggctcct aatgaagctt tcacaatcac ctggcctgag cacagtttgg gtcaggactt

3841 ggtatacttg aaaaaaacat gcaaaaccaa aatcctgtgg ttctggaaaa ggcttcttag

3901 cagaaccccc agacatttac actctgcttt ttcacagggt ccctgaggat tctttggatc

3961 tgggtagttt ggggagcagt attttcaaca agttcatttc gtgctccttc tacaccctgc

4021 ctggatgcta ggccccatct agaatgtgaa caacagaaca aggcagaaca cttgtcctca

4081 aggttctgtt gagtgttaga tccagagaag agacaccccc cacctccccg catcacttac

4141 aggaattctg tttggaaccc aacatcaaat aaggaccgta tccactgtca gaggatggga

4201 agcagcatgt catctgggac attggagaaa ggctcctggg ggaagtggga cttgagctgt

4261 gatctaagta atgaacaact gagagttaaa tcggagagca tcccctatca gggtcctgag

4321 agcaaccagc catggtttaa accagctata aagcctcggg tttataggat agacagtaac

4381 aatggcttgt ctttgggagc caagcagctg gtccaggcat gcagagcatg tctgtatgga

4441 gagctgcctg agagatgctt ttgtttacac ttatcaattg cccatgtcaa agaaggatat

4501 gtacatgaag ttacatcagt atgtaagaga gattttaaca atttttgcag gggaagcttt

4561 catgggggct gatgggaatc taggtaaaca gaaccaaagt ctaaacccaa gatatcccca

4621 gtaccaagac tgaaatgact ctctcctcta tctctagaaa gttccagtga cccaaggagg

4681 caaacacgat gggagtcatt aaagtggggt ggacgtgctg atcatcttcc taattctgct

4741 gcttttgttt tcagccccaa cgtgtgtgct gtgcagaagg ttattggcac taataggaag

4801 tacttcacca actgcaagca gtggtaccaa aggaaaatct gtggcaaatc aacgtgagta

4861 tctgtaacca gccaggagac caagctgtat gcacgctggc tgcagttccc cagggcctgg

4921 gccagccttc tagaaggtca ggttgcctaa aaagccatga agatgcatgt gcgaacatgt

4981 ctgggacctg cgtgctaggg agtggcattt ttaggaagct ggccaatttt gttttgcatt

5041 tttaaggctg ctgacaagac ttggagacat ttttcagggc tggtttgggt ttgcaagaaa

5101 catgaaacac tgcgtgtgtg tgtgtgtgtg tgtgtttctc aatcctcata aaataataca

5161 gatatgcagt ggagaagcca ccagcatgtg actctggaaa agaaagccca ttggtgaatc

5221 tgtactaaag aatgccatcc ctatcttaca gtcctaaggt aaacacccca aaaagactta

5281 gagcactaaa catatgcaga ttatgagaca gcatagcata taatatttgc acagacttcc

5341 tcattcaaac cctagctcta cctgggccag tcgattcatc tttagaaccc tccattgctt

5401 tacctgaaaa gttcgtataa caaaaggacc caccttatgg ggttgttaca aggattgaat

5461 gaaataatgt acataagaga ctgaatatgg tgcccagcat atatcagtgc tcaataaatg

5521 ctagctacta ttattattat caccctagat ttgcaaatct agaccacaca agcagaagta

5581 agagtgccaa cggggtgtgg accagtgtgg ttacaatagg gcttgttgat gtctgtttca

5641 gcaaggaggg aggcagcttt taccccactg cccagctccc tggtggaatc aggtgcatgt

5701 tctaacaatt ctggggaaac ctaatctgtt ttggcactgt caacagatct caaagctggc

5761 tgtctcctat agctaggaag atgtgtatga caaatctcct gagccacttg tgaaggcctg

5821 accttcctcc tgtctccata cataatggga tgattaagaa actctaagcc actctcttaa

5881 gcacttttca atgttaggga tttttaagtt tattgttgtg acattgcttt tgagcagaca

5941 tctcctccaa tttaatagcc aactgaaaga agagaaaatg ctctttcctt aaactgtatg

6001 tcgaaataaa tattccaatg tgtgaccctg attatgttag gcaattagca atcctaatat

6061 gaattgaggg aagttgggat tcatggcaca gctggggaga taccagcagt ccctgggagc

6121 ctgtccaggg caggtccatg gcagcttgct ccatgcctga ttgacagccc agcctgcaag

6181 ctaaaagttg agtgagctag gaggacacac tcccaagatt cagctaacag acacccagcg

6241 atattcttgc tgctatgaac aaaaggagac tatgcaaatt atacaccacc cattcttcca

6301 ggatgcctga cttaaaaaat aagaaaaaag atgggccggg cacagtggct cacgcctgta

6361 atcccaacac tttgggaggc cgaggtgggc ggatcacaag gtcaggagac agagaccatc

6421 ctggctaaca tcgtgaaacc ccgtctctac taaaaaaata caaaaatatt agcgggcgtg

6481 gtggcgggca cctgtagtcc cagctactcg ggaggctgag gcaggagaat ggcgtgaacc

6541 tgggaggcgg agcttgcagt gagccaagat cgtgccactg cagtccagcc tgggtgacag

6601 agtgagacac cgtctcaaaa aaaaaaaaaa aaaaagaaaa gaaaaccttt agtactgatt

6661 gattttttcc catgtgtgta tattatctac tcaaattaac aattaattac ttaattaaac

6721 acaaagccag gcctcaccta attgcttctt ggaaggtgac cagagtgcta gtgccaagca

6781 aacaactctt ctatatctca agagccctgg gcttcagagg gccatctttt ttgttaattc

6841 aagtttctct gaaaatggag acccgtttat gatgacaagc tggctacagg gtagcatctg

6901 ccacactgtt tcgggggtgc cgctgggctg aagcatttgc ccagctagtt aacaatagct

6961 cgataacatt ccctatcagt gtccaggctg agaatactgt cagtgatgag tcgccttggc

7021 tcttgtacct gtatctttgt gtgccaggac aaggcacaag caacagagct gtgtgttgcc

7081 aaaatgttcc tgatgagcag gtcaacccct cgggggcagg tttggatatg ataatgtggt

7141 gatgtggtgg cgcagctccc ttacccagtg agcacaaggg gagtcctcta ggaaaaggaa

7201 gaaatgtctg gatgaggtgg ggagatgggg ttcagagtgg actcaggcaa agcccgatgc

7261 ccagtcccag ctgttggcct agtctcacaa agccagaagg atatgacatt tacattcaac

7321 tcttgaattt gtggccactg ctttgggcaa cttcaaagag agaaaatgaa gatagaaaaa

7381 tattatttga tataaaactt ctaggacaag agaggccctt cctggaacat tacatgtagt

7441 attaggaagg tggagctgcc ctggaaaaga tccagagaac tcagagagag gaagaggtgg

7501 aacccatctc tcttcttgta gagagctcag taagagtggc ttggcagggc tcctgtgtac

7561 ctgagaccaa gaccagtgag gaggctactg tctgaccacc atacggtcag aattcagtgc

7621 catgggtggt caggtgggaa ggggagagga ctgtgctggc tggagttgat gttatcctgg

7681 ggaaagtagg tccctagatg cctttagttg agtgaggagc agactgggaa atgggagcac

7741 agtagtggtt ggggcaaaaa ggactgtctc tgcatgaggt ccataggcag ttggaatttt

7801 ctcagcaaga ctccagagaa ggaggctgga gcagaggtgt atgttgggat gaaaaggagt

7861 aaagtatcat gggggaggag gcagctcagg ttgtcaaggg tcaagaaacc agaaggagaa

7921 tttcaccttg gaagcagaca acgggtacca agcatacagg ggaatacttt gtggtgagag

7981 gtcacacaga gatacaggag ccgacctggt gagacaggag cctggagcca cctgcctgct

8041 tttgtgaggc cccagactcc actgctatca tcaggtgaag ctctgttgcc tgcacacaaa

8101 agcttttctg catttacaaa gagagaaggg cctgagtttc tggtgcaatg cgtcaagctg

8161 acatatggac tttattacag gaagtggtta ccagtgggtc cctatttagt ggctgttatt

8221 gtgaatttta ttgttcggaa attcacttta gcatttattt cagatcctaa atagcaccgg

8281 agtgatacaa tcgctaatca aacaaagagg gctgtgggga gcagacagtc agcatccccc

8341 tctgtgattt caggccctgg tttgattagt agccataaaa ttttttacgt gtggcacttt

8401 gagcaaaggt gcaggaaatt gtggtcagga agcctggctg cctctcgaca ggcttccttt

8461 gtgctagccc cagggagagg aggcctattt aacagccaag tccaagttga catcatggga

8521 ctggaatagt catagcagga gctcagacat cataaacgtg gcatagggag ggctggtgga

8581 ggagctagcg ggtatgggtg gcagctattc attccaaaag tcttgaaatt gtttcacgag

8641 caacacattt cacaagtgcg aagcccttct ctggagccaa gatgagctgg cagagcactc

8701 ctgtttctct agtagcaagt gttcctttgc ccaggggcaa aaatattaat actccttcag

8761 cactgcatta atgcttaaag atttaacttt taaagagatc agctggtgca tggtcgagct

8821 tttccatcag ctggcagggc tttttcagta ggtgtccttc tgggcagggc actggggaca

8881 gctgacgtga aggtgaagaa gagctgtcgt tttcctccct tatatcccac aaccttggtc

8941 ccaagaggaa aaaaaagaag atggtgagaa gtcatccaag cagaccccag acccatacta

9001 gtgcctcctt tcctgtttca tatccctgtg cagccagctg ggatctcttg aataatctgc

9061 tctgggggca ctgagattgg acatacacca aacagcggag atcgaccaaa cgcctctgtt

9121 gggcagtgtt tcctgagggt tctgtcccat tctgtaaact aggaggctga ctagctgaca

9181 aggaatttta ttctgttggg tatttacatg aacctatgtg ccacctgggg taagaccctg

9241 tcgtaggtag aaacatgact tcccaaaaat gtccacatcc taatctctaa ttctgtaaat

9301 atattccctt actggaaaaa gagactttgc aggtgtgatt aaattaagga tcataagagg

9361 gagagattat ccaggattat ttgatgagtc taatataatc atcagggtac ttaaaagagg

9421 gaggcaggct gtgcctggtg gttcacgcct ttaatcccag cactttggga gactgaggcg

9481 agcgggtcac gaggacagga gttggagacc agcctgacca acatggtgaa actccccctc

9541 tagtaaaaaa aaaaatacaa aaattagcca ggcatggtgg tacacacctg taatcccagc

9601 tactcaggag gctgaggcgg gagaattgct tgaacccagg aggcagaggt tgtggtgagc

9661 tgagatcgca ccactgccct ccagcctggg caacagagca agactccatc tcaaaaaaaa

9721 aaaaagaggg aggcagtggg atcagagtca gagaaggcaa cgtgatgatg aaagctgaca

9781 tttgagtgat gcaaccacaa gccaaggaat gcaggcagct tctcaaagct ggaaaggacg

9841 agcaatggat tcttccctac agcctctgtg aggaatgcag cctttgattt taaccccata

9901 aggccgattt ctgactctag cctctggaat tgtaagataa tttgcatgat ctcaagccac

9961 taaatttgtg gtaatttgtc acagaaagca atgggaagcc aacacaggcc ttatttgttg

10021 acttatagat gcatttttct ttatttcaat gtacttttat caatggtctc atgtagggta

10081 ttgctttcaa tgaagatatt aacatagttt caactttaag gtttatatct ggagtttctt

10141 tagaagcttc acaactgacc acttagtaaa cagtaagcat ctgttaagtg cttctcatat

10201 gtaagttcat tcaattctca caatcacact ataagataaa tatgattatt agcccattta

10261 cagatgagga gacaggctca aaagactttt atgcaacctg gtcaaagtca ttcactggta

10321 agctgaggag gtctgtccac ttccttttgc tgcccccagg gggtatcaag cctggcagtt

10381 agtgtcagcg acttaggagg tgaacaagtg agcaggcctg taggacctgg ctaaactgcc

10441 ccaggtctct gtctacagcc tcaaacctgt ggctgtgggt cccagagaca aggcctcctc

10501 agcatcagag aaggatgcct ttgtctcagg gtcatcaacc ttctccaggt tgctcacccc

10561 ctgctgtaaa ggggatcccc aagaccgctc atcagacaag gagcttggga actgaggaga

10621 cacagtcagc ctccaggagt gcccaaaatg ccctcacatg ctgcatacag attgccacaa

10681 ataaagtaca tccacattct gaagactctg tcctcatcac caaccaggct ggcccctggt

10741 gagggctgta gtggttgagg cctttgttgg tagacagtag gttaaagcaa gccatgattt

10801 tctattggga ggcttcagaa tcagctcagc tgtgtttcca agaccaggag ggcagaaagc

10861 aaaccatccc aggcaagcag tccatgggcc atgtcagatg tctagacgtt atgggtctgt

10921 gtttgctctg ccattcctct cggaaactat gatgccctgt atggtttacc ttcagtcaca

10981 ggtgactggc ctacagggcc attccttgtt ccaacgactt ctcgagtata attaatcccc

11041 aggcatttac ggccagagca gccggccaaa tccgtgaagt gcagtggttg ttttaaatta

11101 tattaacttc ttggaaactt attttaggga gagaaaactc agtacttctc tctatccaat

11161 cttgagtaaa aatgttagaa gggactggtg gagagcctcc cagacatccc tacacataga

11221 ctttgggttg acattatctc tttgcacctt ccttgaaact ttcttctaaa ttaggtgcct

11281 tccctaattt aggcaccttc ccagtactag tctgtgacct gttaggaacc aggccacaca

11341 gcaggagttg agtggcaggg agtgagcatt attgcctgag ctccgcctcc tgtcagatca

11401 gcagtggcat tagattctca tagcagtccg aatactattg tgaactgtgc gtgtaaggga

11461 tctagcttgt gcattcctta tgagaatcta atgcccgatg gtctgagatg gaagagtttc

11521 ataccaaaac caccccttcc ccctgccacc atctggggaa atattgtcta ccacgaaact

11581 gatccctggt gccaaaaagg ttggggaccg ctgtcctaag ggatctgctt tttctgacct

11641 gaggtttttc tttattagac tgtatctggc tgaggagaag cctgaagcct ttaatcggaa

11701 cagctttggc tgatgagatt agattcagaa accaacagat tggtcttttc tatgcaggga

11761 agcctaggaa ctggggggct atggctggga agccccctat tgtttccatc ctttcctatg

11821 ttcatcctgg aggaatggca tcagacccat gcctctgtga ttgctcccag cccatccaac

11881 cacagcatct atgttctgcc tgggaccagg gccagggagc atggcacact gagctgagta

11941 taaggagagt ggagcaggcc actgccagcc cagaaaattt tggtcaaagt tgcctgaaat

12001 cttctcagcc ttcgattcac agctgctctc tgctgctctg gggccatgca gaccagttca

12061 gaaaagagtt aatttgttgg ggcagttgga ggcaggtgga ctgccagctt tgacaccttc

12121 ccagcccaca ggctgctgca ctggggctga aggcgtggct aacccctgca cacctagaga

12181 gtgacagaga tcccagactg ggcagcagga aggcaagagg attaagagag agcttcctgg

12241 ctgaaagcca cactcggtta accaggaaaa agcccttggc acgagaagac tcagtggcct

12301 gagggactga gccttggttg ttgggcatgt gctgcataag ccatccatgt gtgacagtag

12361 agtgtagtcc agccactgtg ggacatgggt gctgaaagac cacatggaga ggaacagtga

12421 gtgctgacaa gggctagcct tgatcacttt ggagacaccc cctgtgtctt ctagatgtca

12481 gactttccaa atctgtctgc tatcctccaa acgtgcattt tcaagagcaa tggaaaaagg

12541 attggacttg atggaatgca gcaagagtcc taggtctgtt actacctacc tatgacctta

12601 agaaactcct tcacccctca gaacccttac agctttcttt ctgattctat cctgagttac

12661 tctactccaa gctgagactt ttctgcttag atctatccct tcctcctaaa cccccaacct

12721 ccatttctcc tggtgtcttt ctttacacac ccctcagcat acacacacac ctagccacag

12781 gaaccaatga gttaatattt gaggagttgg ttttcttttg tcctcaatga gatcctggtg

12841 aggccacttg agctgttcag ctcccttgcg gtattttggg gatggaactc agaagccaac

12901 aatatagaaa aagagtcttt ggccagcttt cccaggggct ccatgccata gagagtactg

12961 cacccgtgtg cacagggggc cctgacatga ggactttgag gataacacta ttcctccaac

13021 tctgcttcag catctccatg gattttcaca cagacacttt aggaaagaaa ctaagtttgg

13081 ggggacttga cctaatccca catcacagcc ccagtaatac agccctggaa tttatcacag

13141 aaagcctaga atcccatgca tatcccatgc atatgcatcc ctagtcctat gggttcaagg

13201 cttggagctc tccctggatt tagctgggaa aagttggcag acagttcttc tctgtcttct

13261 agaaatatgg actagaatcg tgagtgtgag attgcaagta acttttaaaa tcatctagtt

13321 taacttcacc ccatttcata gaccaagaaa ctgagaccag agagagaaat ggactttcaa

13381 gttcaccctg ctagttactg atggatcaca agtcaaatct cctgattcta gcactgtttc

13441 tcttacacca caccaccttt gaaagtgtgt caatcaaatc ttactttagt tgcagaggat

13501 gactttagtt tctgaagata aaattgtgag tcaatcaaga tgagtcccaa gacaatagcc

13561 tctttagccc ttataagttc agggatgaaa ggttagaaag aaacaggatg gaaggaggac

13621 tggagaaaaa aacaaaagag gaaggaagga ggaggaagca aacaggaaaa aaaaagaatg

13681 tgcatagctt gtcactcctc agtcatttcc tgggagccca tttctagcaa agtgacagct

13741 gcaactccct ggccacctga gcatcttagc tgatctgtct ctgaaacacc ccctggagaa

13801 cagatgaatc aggcttcatc ttcgcttaac taagtcttcc ctgagacgac tccatttaaa

13861 tgaacaagag caggatttcc tgggcacact gagagcacct tccagaggcc cctccagagc

13921 cctaaagcct gtatttcttc cagtcggcct gtttctttcc tggtgatgtc attaaacgcc

13981 ctttgagagt cccacagtga gcagttctgc ggtaaaaccc gctgcaatta aagtctgagt

14041 cctttcctgt ctcaaagggc atattcatat agaagaaagg aaaaggaagg actggctgtt

14101 tgcatttggt tccaggcctg ttgagtagag gtcgtgctca ctccaccgaa ggtacagggt

14161 agccttcagc agaacctggg gatttggttt taagcaagtc tttcttaggt gtgggctttc

14221 agaacacttc cttccttgca atattatttg aaattctcag tgttttagcc gtccccagaa

14281 tattggttcg ttaaagctgt gtatttcaga tctccagaca gtggtcactg tttgtatatt

14341 ttcaatttca aaccagaaaa caaaagttct tattgattac tttttttatt taaaaaataa

14401 aaagtaagta tcttcgtaag aggagctttg ttttaatttt aaagtttaaa atttgattgt

14461 gaagacagag aaaaacttga tgattgtaga tatattcccc tctttggcta ttcaatcaga

14521 gaactagaaa atcatgagag atttaatgac cactgcctga tacacatatg tcttttacag

14581 atgaggaaac tgagacccag agagatgatg aaattggctg aggatggccc agctggtcag

14641 tgaaagactc agagccagag ctggtgcagg gctctttcta ttccttcctg ttccctttca

14701 ggaacactca ccatcggctt tcctgtgaat aatgttgaga taaaatcctt ggtgcattat

14761 gttttctagt cacaacattg actaggctgc cagagtcctc tgttctccca gttggttggc

14821 tgtaggtgtt ggcagccgcc aggagcattc tacagaacag aggaggagtg agactctcct

14881 tgctcaggaa aggcagacct atgacttagc aaataactcc taagaggaga gtgtttcacc

14941 caccattcct cttccttggc tgtggaggca acttagtgga gaggggccag atgacctgtg

15001 aggaacagtg aagccctgcc taacacaatg tatggttgtc ttgttacaga gtcatcagct

15061 acgagtgctg tcctggatat gaaaaggtcc ctggggagaa gggctgtcca gcaggtgaat

15121 gaatcctccg ggccttgcct gttggtgtgg gtggaaggga atggtgggag agaggagtac

15181 ccacataaaa ggcagcagag tgtgaatggg ggcagtggca caaggacatg gcattctccc

15241 cacgtgccca ctggccccag gctctatgcg aggggctgag gaatggaagc tggaaacagc

15301 gcatttcctg agctgctcct cctggcctcc ttaccacact ggtggagtag actccaactg

15361 tggcctgtcc atgcccttcc cagcaggcac aggctcaggc tcaggctctt ggcctctgcc

15421 tctggctggg agtgattcta aacacatcca gcagggtcag cctgatagcc catcagtttc

15481 cgatcagctc tgctagagag ccgatgggat gtgggaggag ggggtcactg gtgggctggc

15541 aaccccaagc catccccatc tccctctgtg tctaaacttg gccctttgga gttcggtagg

15601 gagaagagcc ataggccagg tgggctcacc cagagtcagc agagagtccc acaaatggtt

15661 gcactgggcg aaagacagca tggcacctgt gaattttatt agagcttttc ttttagtgct

15721 acacacaagt gactgtacag gggagttagt attttgtttt aattttgaaa tagagtcatc

15781 ttttggtatc tgcgggggat tgattctagg acccattcta ggatgccata tcctcagatg

15841 ttcaagtccc tgatataaag tggtatagta tttgcatgta atctatgcat attcttccat

15901 gtactttaaa tcatctcaag attacttata ataccaaata taatgtaaat cctatgtaag

15961 tagttgttat accctctttt aaatttttgt attatctttt attgtatttc aaaaaatatt

16021 tttggtccat gtttagttga atctgtgggt gaagaaccca cagatacgaa gggccaactg

16081 tattggctat ttttttagtt aagaatgtga gactgaggcc aggcgcagtg gctcatgcct

16141 ttgattccag cactttggga ggccaagagg ggacgatcac ctgagccaag aattcgagac

16201 cagcagcccg tccaacatag tgagaccttg tctcttaaag attgtgagac tgggctgggc

16261 acggtggctc acgcctgtaa tcctagcact ttgggaggcc aaggcaggtg gatcaactga

16321 ggtcaggagt ttgagatcag cctggctaac atagtgaaac tctgtctcta ctaaaaatac

16381 aaaaaaatta gctgggtgtg gtggtgggcg cctataatcc cagctactca ggaggctgag

16441 gcaggagaat cgcttgtatc caggaggcgg aggttgcagt gagctgagat agggccgttg

16501 cactccagcc tgggcaagaa gagcaaaact ccatctcaaa aataaataaa taaataaata

16561 aataaatcat gagactgaga cataacagga aggagggcaa tttggttggt tccaaggttc

16621 ctagagtaty tgatgggaga ggttggtgcg ggtggggcca tggaggtact gactcaagtg

16681 gagggacagg tggggaaatg ggatgggaaa agaagattga ccttagaagg ggagctcaac

16741 ctctgaaccc taatttcaga cccttcaaaa tgaatattaa gctcattttg gtctaagaaa

16801 caaaaaacaa atgaacatga aactcatttt ggtcttataa ggtctgagaa accccttcta

16861 aacttcaagc tgctttaaga aataacattt tattacctgc aaatacacac agtactttgg

16921 agatttataa tagtctctta ttctaataga agccattagg gaaccagttt caataaacag

16981 gtaaatctgt aagactagtt tgtaattagg atatctgttt ccagtgtcca ttcctgcctc

17041 tcttatctaa atgtctggga acaagagctg tgctctgctg tctttaaaat gattaaaaat

17101 caccaattag ttgagttcac gtagacaggc atttgactta ttgagttgtt ttaagaagac

17161 tataacaagc cttaagcccc ccagaaacag cctgtctttg ggctttccca catgcctcct

17221 cgtcctctcc acctgtagat gtaccgtgct ctctgtcaga gaagggaggg tgtggttggg

17281 ctggaccccc agaggccatc cctccttctg tcttctgctc ctgcagccct accactctca

17341 aacctttacg agaccctggg agtcgttgga tccaccacca ctcagctgta cacggaccgc

17401 acggagaagc tgaggcctga gatggagggg cccggcagct tcaccatctt cgcccctagc

17461 aacgaggcct gggcctcctt gccagctgtg agatgacctc cgtctgcccg ggggactctt

17521 atggggaact gccttacttc cccgaggggt gggcatgatg aatgggagtc tgcagtcatt

17581 tcctactgtt tcaggaagct ttctccttaa ccccttagaa aaggctgtgg aacttgagct

17641 aaaatatgtc ttaccaggtt gcgtctaatg ccccccgttc cctactgggc agaaagactt

17701 gggtgcttcc tgaggaggga tccttggcag aagagaggcc tgggctcacg agggctgaga

17761 acatgtttcc cagagttgca aggacccatc tcttaaacac agagtctgca gcccctaact

17821 gacaccctgt ccttcctcct aggaagtgct ggactccctg gtcagcaatg tcaacattga

17881 gctgctcaat gccctccgct accatatggt gggcaggcga gtcctgactg atgagctgaa

17941 acacggcatg accctcacct ctatgtacca gaattccaac atccagatcc accactatcc

18001 taatggggta ggggatcccc agccatactg catggccctt ggtgcataat gaacccattt

18061 ctgttccatg tgtgggctgg tttctggggt ttaagctgta gacaacccac cctctttgtg

18121 cctgcttctc cttgggccct ctattccaca gcttgtggaa cccacatttt gctactgtgt

18181 ttgaaaacac tgttttctcc tcccggggct ttgggactat gcctctgttg tgttgactgc

18241 tcatccttgc tgcttctctg ggcagattgt aactgtgaac tgtgcccggc tgctgaaagc

18301 cgaccaccat gcaaccaacg gggtggtgca cctcatcgat aaggtcatct ccaccatcac

18361 caacaacatc cagcagatca ttgagatcga ggacaccttt gagacccttc gggtaaggga

18421 ctgccctggg tggaggccca ggcttgggac acattgcctc ccaagagggg cctagcagga

18481 actcttctgc aggagaggta gaggatggct cctgtagggg aacatagagc aggttcccct

18541 gaatgccctt gaacatggag aattcattga ccagacattc agcttgacct aacctgtgaa

18601 attctccatc ttctttataa agtgttccct tccttgcctc ccctggaaag gtcagtggtg

18661 tgtggctgca gcagcacagt gtcctctgag ccctggacct gcactgtggc ttccagaggt

18721 ggcagttccc acatggggta ctagaataaa tggcctatca ggctgtgtgt gctttgggat

18781 cacatgtccc caccctagga ccctggttcc aaccatacgc atgttctctt ggagcccaga

18841 acagcagaga agccaccagt gtggacacag aagtcaaggg tctgatttcc agcctggctt

18901 ctgactgctc tggggccgca ggaatacggt tccttccccc atgcccagca ggcatttgtc

18961 ttacaactgg aggggaaggc atgttcctct tggcaaggac tgctcaggag gaagtggagg

19021 caggctgccc tgtcagggtt tttgccttga ttcaaggaga acttcctaac cacaaaggat

19081 acaagtggga gtgaggcgga ccctccctag agatctccaa cacagagaga caaacacgct

19141 ggggctggct ggcactgaca ggcctcgcag gtgtggatgg ctgttagctg ggagcttcgc

19201 tgtctaagct cctctcccat gcttttcttc tgggttgctc gaaggacggg ggtctgcaag

19261 aaaatgatgt tcccacatag ttggcagcac gtgaacagca attgatccct ttgcatcacc

19321 tcctcttact gtttagattt ggtaaatatt tcttccttcc ctcttctgac cctccatttt

19381 gccgatcttt ccttcttata acacatactt actaggtacc tgctacttcc cgggtgggcc

19441 tatgtgccag gagtatagag gtgaacaagg aaggcaaagt tctattctca gtagagctaa

19501 tactctatct ggagagagac aacaaacaaa tcaacaaggt agccaggggc tgtgataatt

19561 tatgtcaagt gggcaggtaa atcgggagtg acagtagtgc agggaggatt ggaaagtcag

19621 ggagttctct ctggaggagg tggcttttga tctgcagcct aaaggatgag aatgggtcca

19681 ttatacaaaa tgctggggca agagcacacc cagtagaggg gagagtaata gcaaaggctc

19741 agggcaggaa gggcaaggga gaggccagtg ggtgaggtca catgtgaagg gcatacaatg

19801 ggcaaagaca aggccagagt ggccaggccc aatcctccag gacttgcaga cctgggaaag

19861 agtgcatctc catcctggga gcagcaggaa accactcagg cctttagaag atccttctgg

19921 cagctgtgta gagaatgggt ggtgtgatcc ttccatgcat gggctcatgt acgtgattac

19981 cagtaactgt cgagtgacag tgtgaggagg gctgcaagcc atgagtgtag gcacagcaga

20041 cagactcacc tttgtctggc ggtgagatgg ggtgggaagt gtgccaagtt gacctcccaa

20101 agaaatgata ttttagtgga agaatgaata gaatcagaga agcaaagtaa gagggaagag

20161 cagagaggac agcagggaca aggacttggg ggcaggaaga ggaaaggcag gttaaggaca

20221 tgaaagatgg ccaggctggc tggagctcag gcccagcaag gccccctggg ggccatggtc

20281 atgggtgagc ttgggtttgg cttctgtttt cgtcttgggc ttctgtgaaa gcctcgagcc

20341 cttgcgggga accagtgaag ctgtgtgtgc atcttctgtg gggagtgcca gagtcttcag

20401 ggagcactcc atcttctctc ctccccacag gctgctgtgg ctgcatcagg gctcaacacg

20461 atgcttgaag gtaacggcca gtacacgctt ttggccccga ccaatgaggc cttcgagaag

20521 atccctagtg agactttgaa ccgtatcctg ggcgacccag aagccctgag aggtgagcat

20581 cctttggctc ctgctgctgc ctcatttgtg cagctagatt gagcccaaga cctgctctgg

20641 tccaagatga acataccacc tgccatgagg tgaccctcag gatatccact gcagccatgg

20701 gctggggtca tcctgtcctg ttgcttcagc taaccgtgtc tctagcagcc acactactct

20761 gagggctgac tacagaatcc agcagctttt gtctgggaga gctggactga agagaggcat

20821 agctggagac ccatagctgg ccctggccag aaacagggag agtgaaaggc tggaatagcc

20881 aaggccagag caaggctaat aggtagagca acagcttaca ggtgtggggg tggcagatac

20941 tcgcaccctt gaaatggatt cctcatgccc acgcttcact attcttctct gtggctaggg

21001 gatttatgga taaaccaaaa ttacagttaa aaaccagcca taggccaggc acagtgactc

21061 acgcctttaa tatcagcact ttgggaggac aaggtgggcg gatcacctga gatctggaat

21121 ttgagaccag cctggccaac atggcgaaac cccatctcta ctaaaaatac aaaaattagc

21181 tgggcatggt ggtgggcacc tgtaatccca gttactcagg ggctgaggca ggagaaccac

21241 ttgaacccag gaggtggagg ttgcagtgag ccaagcttgc accactgcac tccagcctgg

21301 gtgacacagc gacactccgt ctcaagaaaa aaaaaaaaaa aaacagttat agtagtcaac

21361 ttttgactct ccatttcaga tttcgtcatg ccctcctcaa tgagctgcta agttaggcag

21421 tgcattgatt attgctgcag gagagggaag gaaggagcta acgtgttttc acatgttttc

21481 cttttggaga tgagaaagga ggactctgcc ttccccctac cctgcccctt tctactccag

21541 gacctctgaa aggccatgag cacaaagctg ctgcctgagt cccctgaaat gcagggtacg

21601 ccccaggtct ctgatgtacc ccaccacact tttcctctca aacatattcc aggatcactt

21661 gatttctttt gaatctattt aaacccaccg tgtcaatgtg ctatataaaa tgtctaatgc

21721 atttcagaca ccctatacat ctatacattt aaagtgttct ccttctatct gtgcagggat

21781 gggaaagggc atatttctga aagcacagat gggaagacgg gatttgttcc gtgtccaggt

21841 gattatggta cctctatgcg cctggccggc actggggaca gaggccatga aaatgaatac

21901 agcacagcct ttgcctccaa gaaacttaag acctagtaga aatggcaggc tttaaaacag

21961 gttgttggga tctgatttgg tgagtgcaat gacagagata ctcacagcac aaaatgggga

22021 atgagggcgg gcattgggac acacatagcc ttaaggggcc caaaggcttt tagaactgta

22081 ttccctatta aaacatgatt tgcacagagc acattctttg ctttggagac ctcagaactc

22141 cttactatag gccgggcatg gttataatcc cagcactttg ggaagccaag gcgggcagat

22201 cacttgaggc tgagagttca agaccagcct ggccaacatg gtaaaacccc gtctctacta

22261 aaaatacaaa aattagctgg gtgtggtggt ggccacctgt aatcccagct actcaggagg

22321 ctgaggtagg agaatcactt gaacctggga ggcagaagtt gcaataagcc cagatcatgc

22381 cactgcactc cagcctgggc aacaaagcta gactctctca aaagaaaaaa acaaaacaaa

22441 acaaaacaaa acaaaaaaaa ctccttatta taaactgtaa gaaaaaaaag gcccctactt

22501 cgtccctttt gcaaatctgc cttttcctac tcactaacca gctggttcag agcaaggaca

22561 ctctgtttgg tgccatcgct gcagactgga aggaagaggt ccttgcccca cacccaacag

22621 tctcctgctg ttaccggcag gttggcaggc aggcaggcga gaagcagcca gggctggtgg

22681 tgtgtccagt ttgaagacta gtttccagcc ctggccctgc tcaccctcca agtggccctg

22741 gcaggttcct ctaccacatc gtggacttca ccttccttct ctaagaagct caatccccaa

22801 ggcctcattc ccataggcct tctcaccctt tttctttccc tctggctgaa tgtggccagc

22861 acgggcttcc aaggccatca actcgtctgc agcagcccca tgccttgcag ggcctcagag

22921 cttcctcctg cctatgacag tgtggttttg gttcccacac ttgggatcag attgaaactc

22981 gcctccgtgg tgagaatatg ggacatagag cctcggtgac cttggtgagc agcagtccag

23041 gccacctgct cagcctgggg ttgggggggg ctcctcctcc ttgactggtc cttgcatttg

23101 cctccatcca gcctgtctgg gctctccgag gcaatggaga ccagcaggag tcacgatggg

23161 tcaggagccc cctttgggcc tcagccctgc cctgccccct aaagtagcac ttggataagc

23221 aaataaatta ttatacttac tatttatggg tgtggtgaat gggatggcaa aggccaagtc

23281 ttactgatca ccaaacctta agatatatcc tggcagctag tagacccttg ggctaaatga

23341 acagaaaact ggacaaataa agtgtacaca aataactcaa agctgtcatt tgtacacttt

23401 tcgtcttttc ctactacagt ttacattttt ataaaggtga gtagatttct aaaatcccgt

23461 ggtaggctct cttgagtttt tcttgtatcc ctgaagttca gctacaaata agctaatcac

23521 taacatttgt tgagcattta ctctgttgtc aggccccgtg ccgagtgctt taggttcaga

23581 atttcatgtc atccccacag cagccctagg agatgaatgc aattcttatg tccacttgac

23641 tgataaggaa gttgaggttc aaagaggcta aatgactctc ccagggtccc acagctggaa

23701 agtggccaca gggccccagc tggttttcta gggcagcagg cagaaggcga ggaggatctg

23761 ggccctgtgg tgccccagcc tcatctgagg gtcctcatct gagagaacag gatcctcaca

23821 gcatgggcag gctgcaagtg gtccctgagg ttatcgtgga gtggaccctg acttgacctg

23881 agtctgtttg gaccccagac ctgctgaaca accacatctt gaagtcagct atgtgtgctg

23941 aagccatcgt tgcggggctg tctgtagaga ccctggaggg cacgacactg gaggtgggct

24001 gcagcgggga catgctcact atcaacggga aggcgatcat ctccaataaa gacatcctag

24061 ccaccaacgg ggtgatccac tacattgatg agctactcat cccagactca ggtaggccag

24121 gcctccgggg gccttggccc tgcctggccc accatctctt ctgccatcct ttgtggcggg

24181 ggaggggaaa ttcagagatc tttgggcgac ttccctgcct ggacccagct cacagcttct

24241 cggccactgc aaatgtgtgg gttgtgacca gactgatgtg tcttgagctt caggcttgca

24301 agtgcagtgg agaggcagtg gggagctatt gaaggggtct ggggacagac tcaatcacag

24361 aggcctttca gaagatctgc ctgctgtgca tgggcaaaga gggccacttg ctgacctcag

24421 agcatgtgct ttctcagtag tgcccaagct gtcccatggt cactgaccca gttagaatga

24481 ctgaatggac tttggcttgt gtctcattag gaatcctagc cccattctag tcttccagtg

24541 agatctgtcc atgagtgaag gaatctcaca ggaaaaaaca aaatgcttct atgggtgtgg

24601 ttgctggcct tatctacacc acagaagcca tcacacagac tgtctttctt cccattgtta

24661 gaatgtgccc tgaccaagca gcccacaggg cctgggacag aggctgatct ctgcctaact

24721 gagctcacct ctcctccctc tcctcctgac tggttagatt ttctaggtga ctgttcccct

24781 gatgacacaa gcccgctggg ccccagcagt gtttagaggg gttgttgact cacgagatga

24841 cattcctgct gatgtgtgtc atgccctggg gtggatgaat gataaatgaa aacagcgctt

24901 ttaacttttg aacccacttt ctccttcctt gtagccaaga cactatttga attggctgca

24961 gagtctgatg tgtccacagc cattgacctt ttcagacaag ccggcctcgg caatcatctc

25021 tctggaagtg agcggttgac cctcctggct cccctgaatt ctgtattcaa aggtaacatg

25081 gggaaggcat ccctgttaga ttgtccctgg aggcagcttc cccacccctg tcacctccac

25141 aacactctcc gatttacagc accccatggg acattagaac ttccactcag ctcaaccaaa

25201 agcagatgtg acttcagcag aaacttcaga ggctctgttg tttcattagg cagtgcagag

25261 aatgcctttg gggagccgtt cctcagaact caagacttga catctgggag gcagccgttc

25321 ctcagaactc aagacttgac atctgggaga gcagagcatt cccttgcctt tctatttgca

25381 gggtcacttg ccaatgtata gtcaagaggt cagagtgagg gtacagctga gctgcagccc

25441 caggaaggca gagaaggggg ccaagttgtg tgcgtgcctg cccttccctc ttagggcaaa

25501 actccaaaca cccttgatta tctggatctt ctttaattct ccatagaaga taccagatgt

25561 taaggaatat tcgcagcttc acttggtttc tcaatccctg tttccaaact caaggaggga

25621 tgggcttttt cactgtattt atctctcatc actctcttca ttgcaggagc acatctctct

25681 ggacctaacc atcacccttt cttgtagatg gaacccctcc aattgatgcc catacaagga

25741 atttgcttcg gaaccacata attaaagacc agctggcctc taagtatctg taccatggac

25801 agaccctgga aactctgggc ggcaaaaaac tgagagtttt ttttatcgt aatgtaagtt

25861 ctgggtccta aatcatgctc ctgggaagct ccttactgtg ggacttgtat tagtgtaaaa

25921 aaaaatgtcc tcaataagca ggagtttgca tgagaactgg ttgctgacaa ggaaggaaat

25981 aatttctgga aaatatagat aacaaaatga gatcctgcag aaggattgga atctcttttt

26041 ctggaggcct ttgagaataa accacacaat tatccaacct gtattgtgaa ggaataagtc

26101 cttcttgaat tcaggaatta acacctggga ggagggatgg agttcagact ctttctgagc

26161 ttatgagaag agaagccccc taaactaaaa tacagccctc cttggtccaa aaggtgcctt

26221 ctctcttctg ctgtatcttc tttgttttca aacccaacag ttaccctgga aatcaaaaag

26281 gaagtacaac tcaacatagc tcttgcctgg gaccaaccag caccatttgg ctaaagatgg

26341 ttatcatctg ttaaacaaag aaataaataa atgggttcaa cgtatttatt tcaacattgt

26401 caatggacct catgtgtaac tgatattctc attatgggac ctctgtgtga ctttattggg

26461 gcctctctaa ccgttctttc cttaaggaag accatttatt gttttatttc ctggagaaaa

26521 tacatcattt tatcccagcc ttaataaccc atcccagtgt atactccttc atcttcatgg

26581 ataatgaccc tcctacatgc tctgaacaaa tcaggaggcc cctcgtggaa gtataaccag

26641 tcctttcttt ctctgtccct cttctgtgca gagcctctgc attgagaaca gctgcatcgc

26701 ggcccacgac aagaggggga ggtacgggac cctgttcacg atggaccggg tgctgacccc

26761 cccaatgggg actgtcatgg atgtcctgaa gggagacaat cgctttaggt aattagttcc

26821 atccccgggt ggagcttctg cccagtggtc atgctggagt gggatgtggg gccccagcta

26881 tttgtcaagc tttcttctac cttggggatt caattaacac tagcagtgca ctgctgcgac

26941 cttccagact tgggatgggg aaaaggcaag ggtcgccttg aaagcttaca ttgggaagaa

27001 gggttacttc taagagtgta atcttcacat gcatgggaag cagggagggg ggactacatt

27061 tttatgactg aagtgcaagg aaaacatcac cctctcattg taaagctcca agtgagccaa

27121 gagcacatag tttacagtgc acgatgagcc tctcactctc tgcgcagtat ctgtttattg

27181 caactgaagc acccttgtga gtttgtttt ttgcccggct atctccattt ctgacttgct

27241 cattcacctt ggggtgctgt catattgaat gtttccctgt cactgacttc agccacctgc

27301 acaagggctt ggagaccaca cccctctgcc ctcccagaat catatccctg gaggctcagc

27361 tagtctctgg gtcagccata cctctgccct ttcttttccc tcctttctcc tgtggcctct

27421 gacgtctggc catttaacag agcttagcat ttttgctggg tggagagagc tggagcctgg

27481 aatcactccc tctttgtgca tacggagggc atgaaaacca aggtgtgtgc attccagtgg

27541 cctggactct actatcctca gtggtgaggt atttaaggaa aatacctctc agcgtggtga

27601 ggtatttaag gaaaatacct gttgacaggt gacattttct gtgtgtgtat ctacagcatg

27661 ctggtagctg ccatccagtc tgcaggactg acggagaccc tcaaccggga aggagtctac

27721 acagtctttg ctcccacaaa tgaagccttc cgagccctgc caccaagaga acggagcaga

27781 ctcttgggta aagaccaact taagtacacg tctccatttt tctaaagtag tgatccctca

27841 gggccccagc agcaaacagt tcgcacatca aggattgact tgaagggatt ttatgacaag

27901 actattagtg aaagagtggg cgggactaaa ggaactagca aaggatgagg ccaaccaggg

27961 actagcaacc ctgggaagcc tttactaccc ctaggcctgg gggaatggga ggatgagagc

28021 aggaaccagg gaggtcatga gccttggaca agggcacaga acagcagcca gagccatgtg

28081 cagccagcca ctgtcagaac catgcaaggg ggaccactca gcgccccagc ctccctctca

28141 gacagttgcc atctgggtct cttgttggct gatgcgagag caggagggag cccactgatg

28201 cagttcatag agctcagcct cctgggcagg aaaccgggca gagaggagta gaaaagaatt

28261 aagggtggct gcgaccagcc cagtcactga ggcacgtttc ccactggaga cctatgagca

28321 cagtgataat aaagccagtt acctgcactg actatccctc cagacaaaag ctttcccaag

28381 aagttagtca tggctctgag agatctagtt gaggatgttt ggcaggggat ctagtggtta

28441 cgggtggcta agaaaaatga ggaaggtaag agtatcttgc agcctgtgtt gggaggatta

28501 aataggatgc cacacacagg gccaggcaga cagcctggtc agtaatagcc atgacgatgg

28561 gggcgggggg agcaggaatg ggagttgcag tctttagctc agatgcatgc ctgtgagaga

28621 tgcttccact ctcacagaaa gatgagacca aggaaaagga ggaggaagag gaaggacctt

28681 gacaaacctt ggggcccaca ttgtctacac ctcccttcct gctctagagc agaatagaaa

28741 gttcaggttg caggcagctc taagttgaat tcgtgtcctg tttaattttc tttattgcta

28801 aatgaatgcc tgtgtctgtg atgctgacgt atgttcctaa ggagagggga gaagttcatt

28861 ctgaacataa acttttcatc ctctctctgt ccagcaagaa tggaatattc cccaagtggc

28921 ctgagccagc ttggctttct ttttgttttc aattatgtgg gagttgagga gggggatggg

28981 aaaagcttcc caaacacacc ctcccccagg cctgaggcac ccctggggga cagagagtgt

29041 tagaggttgg tacaggtgtt agagatattg aaaggacatc ccatgcaccc caggggctgg

29101 tgtggctctg tacttccagg caatattttg tggaagggga accttgtcag ctccaggttg

29161 tggatgtttg aaaatcagtt ggtacccagt ggctccatcc tctggcaggc atgtggattt

29221 gtcaataacc aagtgaactc tccaaaataa gttaaaactt cctcccttct cagtttcaag

29281 atgctggaaa tagctgttca taagccctgg ggaaatttag ccctttggct ggtaatggga

29341 gtatccgaga tgagagggca gctggaaact ttcggaatga cctcccacac ttaatttggg

29401 aaatgcctct gcacctttat gggcaaccag atgcctgccc cagttgctgg agacactgat

29461 gtgggctgaa aggaatgctg agacgtgacg aggagagatg ctgcggaggg aatatccccc

29521 tcagccctga cctcatcggc tccatggctc ctccacagta cagctgtcta ctcttttaag

29581 ttctcccttc aggaaatagc catctcaaac agaatgtgca tttgagggca gaatgtgtaa

29641 atattgcact actgtgttat aaccgtcagg agccatgctg atgatgaaac gtcccagatg

29701 ccggtgctgg aaaggtccct ggctttccaa gcaaatattt atctcatgga aacatgagtc

29761 atactcacag aggagtatgg attaactcct tctcagcagc cagggagccc agcatcccag

29821 acagcatatt taacccagag gccaactgac tgctggggca gatttgtggt catgaacatg

29881 tgctttgtgt cctctgacca ttagacagat tgtgggtcac aacgttgagt atacagtggg

29941 agcttaataa gtgcttattc cctgggcagg gagttcttca tttcaggggt gaccacttac

30001 atcttctcct ctgggccctc cttgaccagg ctaattacca ttcttgggat taactctatc

30061 tccttttccc gcaacctgca ggagatgcca aggaacttgc caacatcctg aaataccaca

30121 ttggtgatga aatcctggtt agcggaggca tcggggccct ggtgcggcta aagtctctcc

30181 aaggtgacaa gctggaagtc agcttggtaa gtgtcctgca aatcaaaggc tggctaaatt

30241 tccccagggc agggctccag gacatatctc acccccagga tggaattata cacacacaac

30301 cttcaagttg cagcccgaat ctctgagtgt aattcgtcca aagaaaaaga gaaaagagaa

30361 gagggtcttc agggaaatca agtgagatca tagttagaca tgagtaagaa cttccagatt

30421 tacaagggaa tagagcatct gatttggcat ctgagagagg ctattagatc ttccttctct

30481 taaggaggtt gtaggcaact agttatgtga ctgaagagat cagtctgtac tcacaccatc

30541 ccacccccca aacccagggc ttcactgagt tgtaccatga accagaccat cccaagaggc

30601 tttttgagtt ctgacacttg ctctgtgagc cttcccttgc tctgcacatt gatgatataa

30661 ctttgtaact gcactaagag tgttcctaaa gcagatagcc agccgagctc cagaaatctc

30721 cctggctgca cctgcagagg ccactgaccc ctctgtggag ggaccgctct tcagtgtgtg

30781 gctggcttct actctctgct cctctctctt ggtcttcagc catccattgc tcaccagttt

30841 ctcacgagga gcataggaag atatgcatgt agggaggtag gcacggggat gacttgtttg

30901 actttagcag gtcattcaag aatctcctcg cacctggttt cagatgctgg ggtcctgtct

30961 gtcacaggct tctgtgcctc ctaccccctt gagtttgtca catggccctt caggaaggcc

31021 tgagatagat ttgccctggg tgggcctcct atgagaaaat cttaagtgag gcacccaggc

31081 aaaatggaaa gagccttttg cccagagcag gaagcctgtc ttccatttcc agctgttcca

31141 cctacttagc ttaaaagagg cacttcgcct gtcttcagtc tcagtctcag tctcctcttc

31201 tgtggaatgg gacaataata tctactctcc ttatcataca ctgctgtgag gactgagtgg

31261 atcacacaaa aaagcattat gtaaattgca aagtgctaaa tccacacagg agatttgaat

31321 taatccacca cactgaaggt ctgtcaaggg cagggactgt ttcattcacc agagtatccc

31381 cagtctaaca caggacttgg catatgaaaa gtgttcagta ggccgggtgc agtggctcat

31441 gcctgtaatc ccagcacttt gggaggccaa agtgggcgga tcatctgagg tcaggagttc

31501 aagtccagcc tcgccaacgt ggtgaaacca catctctact aaaaatacaa aattagctgg

31561 gcgtggtggc acatgcctgt aatcacagct actctggagg ctgaggcagg agaatcactt

31621 gaacccagga ggcggaggtt gcagtgagtc gagatcatgc cactgcactc cagcctgggc

31681 gacaagattg aaactccatc tcaaaaacaa agaacaagga aaaaaacgaa aactgttcag

31741 taaacacttg ctgaatgaat aaaataaata tataaatgta taaataaatg ctctactttc

31801 aaccactact ctgtttttct tttagaaaaa caatgtggtg agtgtcaaca aggagcctgt

31861 tgccgagcct gacatcatgg ccacaaatgg cgtggtccat gtcatcacca atgttctgca

31921 gcctccaggt aagtgtcgca tccccactga ctctgcagcc agtccttttc ttcatgtggc

31981 agttggtgga gagaagaaaa actgttctaa acaatgatga gaataacatg taattgtgat

32041 agttaaactg tgcctatgtg actgattgca gagtgaattg ggagctgttg gttttgaatg

32101 caccacacta aggaatgtga ggacacattg ctctttgcgg agttgcccag ctatattagc

32161 tcccctcgga cacagcccag ttttctgtat tcgcgtggat gctgtccgcg cgattcccag

32221 cactcctctt acagcatctc acctcagtgt atgttccttg cctccagtgc agttgaacct

32281 cagtcctgcc tctcctcatg tgtgcattca cctttcttgg tgctctctcc ccatgggcca

32341 agttctacca tgagttatga aacattatgg agaaaacatg tctttggaaa tgtgagccag

32401 aaagcccacc agtgcccctc agtcacggtt gttatgaatg acatgctaat ggtttcactc

32461 tcgtcaaacc tgccttttct ttcctcttca gccaacagac ctcaggaaag aggggatgaa

32521 cttgcagact ctgcgcttga gatcttcaaa caagcatcag cgttttccag ggtaagatgc

32581 ctgctaggtt tgcgcctagc ctgagcagcc tcaggtcctc tgtttgggcc atagaggagc

32641 ctctccagcc cctgtcttcc ttggctgctc cccagggctc tcttaaaact tctccccact

32701 cccactgagg catcctcagc cccagcctgt gtcaaattca gagtaaagaa ccaaggcaac

32761 tccctggctt tcatgggcca aagcgcaggc tttcacaccg aggcctctga gcctcagatc

32821 atggggaagt cactgctgga gagaacagac atagctctgg aagccatctg cccaagaggg

32881 cagcccatcc caagttcatc ttacagtggc caggcctgcc ctgagccggg gcctctgggt

32941 cactcttctg ctgtccatgg cattgcccat cctgggtgag gctggggctc tcctgggcac

33001 tgtatgtatt ctggatacag ggatactggg ctcgctatgt gtgtggagcc atcccttcct

33061 tgccccagcc ccacctccct ctcaaaccct ctctggctct ttctgagctt cctttcctgc

33121 tccccagctt gcccagtgct cagtgcccca cttggctctt ttgctacttc gggtcaggtg

33181 gagcctcttg ggaatgtgaa gtgccttaca gaaagattgc acttcaagag gagaggctgc

33241 agggagccat cctaaaccca gaggcctgga gcttactgtg tcactttact tttgtacaca

33301 ggggtctcct tagtgccctc gagaaggatt cttggccctg agcttctact cctgaggcca

33361 cctctgtgca gccccagctc cctcaactct aggctgtagt ctcagtggga aagcctggct

33421 tgggggtctc ctaggaatgt ccacctgaag gcacacttga taggggcttg cacaacttat

33481 gtctgccaag gccacctgag gaactccctg gtgcctataa gttccacctt ccccttcctc

33541 ttcctcgccc cagcattttt tctgagtagg ggtggcaatg ggcaaagcca ttgtcataag

33601 cagttgcagg tataactttc actagaaaac ctgacacctt gtgttttctt tcaggcttcc

33661 cagaggtctg tgcgactagg tgagtctggt ctgggtttga agtcattgca gacctgttta

33721 ggccttaccc ccaagcaagc ccaagcctgc catctgctgt atatagataa gaacatcatg

33781 gtgcagtaaa agaagcctgg cctttggagt cagaacagca gggtgacttg gggtcagacc

33841 cagagcaccc catttccttc tctgtaagat gaggataata agagtaacaa ccttttaggg

33901 ttaaggtgag ttttcagctt aggaagtctg ggaatattgc aaagggcttg gcaggaaccc

33961 atggtgagga tctagttcca agttgatagg tacagaaaac cagaacatcg ggccttgagt

34021 aaagagtgaa gtttcacaaa ccacaaagca cctgctatgt gcaggagagc atggcagaag

34081 gaggctgctt ggccctggtc cttgagattc tgacagtgtc ctagacagac atggggagat

34141 ctgcacctat ttgacgttac caacttctct ttttcagccc ctgtctatca aaagttatta

34201 gagaggatga agcattagct tgaagcacta caggaggaat gcaccacggc agctctccgc

34261 caatttctct cagatttcca cagagactgt ttgaatgttt tcaaaaccaa gtatcacact

34321 ttaatgtaca tgggccgcac cataatgaga tgtgagcctt gtgcatgtgg gggaggaggg

34381 agagagatgt actttttaaa tcatgttccc cctaaacatg gctgttaacc cactgcatgc

34441 agaaacttgg atgtcactgc ctgacattca cttccagaga ggacctatcc caaatgtgga

34501 attgactgcc tatgccaagt ccctggaaaa ggagcttcag tattgtgggg ctcataaaac

34801 aagaaacata caatccagcc tcatgggaag tcctggcaca gtttttgtaa agcccttgca

34561 atgaatcaag aatggcatca ttataagcta tgagttgaaa tgttctgtca aatgtgtctc

34621 cagctggaga gtggcttgga ggcttttatg gggccctgtc caggtagaaa agaaatggta

34681 acatctacac agatttccct attgtgacag agccatggtg tgtttgtaat aataaaacca

34741 tgtagagctt

TGFβI gene protein product (βIG-H3 protein sequence;
NCBI Reference number NG 012646.1):
MALFVRLLALALALALGPAATLAGPAKSPYQLVLQHSRLRGRQH

GPNVCAVQKVIGTNRKYFTNCKQWYQRKICGKSTVISYECCPGYEKVPGEKGCPAALP

LSNLYETLGVVGSTTTQLYTDRTEKLRPEMEGPGSFTIFAPSNEAWASLPAEVLDSLV

SNVNIELLNALRYHMVGRRVLTDELKHGMTLTSMYQNSNIQIHHYPNGIVTVNCARLL

KADHHATNGVVHLIDKVISTITNNIQQIIEIEDTFETLRAAVAASGLNTMLEGNGQYT

LLAPTNEAFEKIPSETLNRILGDPEALRDLLNNHILKSAMCAEAIVAGLSVETLEGTT

LEVGCSGDMLTINGKAIISNKDILATNGVIHYIDELLIPDSAKTLFELAAESDVSTAI

DLFRQAGLGNHLSGSERLTLLAPLNSVFKDGTPPIDAHTRNLLRNHIIKDQLASKYLY

HGQTLETLGGKKLRVFVYRNSLCIENSCIAAHDKRGRYGTLFTMDRVLTPPMGTVMDV

LKGDNRFSMLVAAIQSAGLTETLNREGVYTVFAPTNEAFRALPPRERSRLLGDAKELA

NILKYHIGDEILVSGGIGALVRLKSLQGDKLEVSLKNNVVSVNKEPVAEPDIMATNGV

VHVITNVLQPPANRPQERGDELADSALEIFKQASAFSRASQRSVRLAPVYQKLLERMK

H

Claims

1.-19. (canceled)

20. A reaction mixture for detecting corneal dystrophy in a human subject, the reaction mixture comprising:

(A) at least a first amplification primer pair for amplifying and determining a first TGFβI gene sequence from a biological sample from the subject; and

(B) a first set of at least three detection probes including a first detection probe comprising the sequence of SEQ ID NO:45 or 47, a second detection probe comprising the sequence of SEQ ID NO:46 and a third detection probe comprising the sequence of SEQ ID NO:50.

21. The reaction mixture according to claim 20, further comprising a second set of detection probes including a fourth detection probe comprising the sequence of SEQ ID NO:25 and one or two detection probes selected from a group consisting of a fifth detection probe comprising the sequence of SEQ ID NO:26, a sixth detection probe comprising the sequence of SEQ ID NO:48 and a seventh detection probe comprising the sequence of SEQ ID NO:49, and at least a second amplification primer pair for amplifying and determining a second TGFβI gene sequence from the biological sample from the subject.

22. The reaction mixture according to claim 20, 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:43, and the second amplification primer comprises nucleotide sequence SEQ ID NO:44.

23. The reaction mixture according to claim 20, wherein the first TGFβI gene sequence comprises nucleotides encoding amino acid residue 555 from the biological sample from the subject.

24. The reaction mixture according to claim 21, wherein the first TGFβI gene sequence comprises nucleotides encoding amino acid residue 555 from the biological sample from the subject and the second TGFβI gene sequence comprises nucleotides encoding amino acid residue 124 from the biological sample from the subject.

25. The reaction mixture according to claim 24, 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:43, and the second amplification primer comprises nucleotide sequence SEQ ID NO:44, and the second amplification primer pair comprises a third amplification primer and a fourth amplification primer, the third amplification primer comprises a nucleotide sequence SEQ ID NO:1, and the fourth amplification primer comprises a nucleotide sequence SEQ ID NO:2.

26. A method for detecting whether or not a human subject carries a mutation that causes corneal dystrophy comprising:

(A) amplifying a first TGFβI gene sequence from a biological sample from the subject using a reaction mixture comprising at least a first amplification primer pair for amplifying a TGFβI gene sequence and a set of at least three detection probes including a first detection probe comprising the sequence of SEQ ID NO:45 or 47, a second detection probe comprising the sequence of SEQ ID NO:46 and a third detection probe comprising the sequence of SEQ ID NO:50;

(B) hybridizing one detection probe of the set of at least three detection probes to the first TGFβI gene sequence; and

(C) detecting whether or not a mutation in the first TGFβI gene sequence based on (i) the hybridization of the one detection probe of the set of at least three detection probes that has hybridized in step (B) to the first TGFβI gene sequence and (ii) the failure of the other two detection probes of the set of at least three detection probes to hybridize to the first TGFβI gene sequence.

27. The method according to claim 26, wherein the reaction mixture comprises:

(B) a first set of at least three detection probes including a first detection probe comprising the sequence of SEQ ID NO:45 or 47, a second detection probe comprising the sequence of SEQ ID NO:46 and a third detection probe comprising the sequence of SEQ ID NO:5

28. A method for detecting whether or not a human subject carries a genomic mutation associated with corneal dystrophy in a sample from the subject, the method comprising:

(A) using epithelial cells of the subject adhered to a tip of a substrate;

(B) agitating the tip of the substrate in a lysis solution that lyses cells adhered to the substrate;

(C) removing the substrate from the lysis solution upon completion of the agitating (B);

(D) incubating the lysis solution after the removing (C);

(E) isolating genomic DNA from the lysis solution to form a gDNA solution; and

(F) determining an identity of at least a nucleotide present in the TGFβI gene using at least a first primer pair for amplifying a TGFβI gene sequence, a first set of at least three detection probes including a first detection probe comprising the sequence of SEQ ID NO:45 or 47, a second detection probe comprising the sequence of SEQ ID NO:46 and a third detection probe comprising the sequence of SEQ ID NO:50, and the gDNA solution by concurrently exposing the gDNA solution to the first set of at least three detection probes, wherein:

the at least a nucleotide is located at a particular position of the TGFβI gene corresponding to a single nucleotide polymorphism (SNP) associated with corneal dystrophy.

29. The method according to claim 28, further comprising:

(G) determining an identity of at least a second nucleotide present in the TGFβI gene using at least a second primer pair, a second set of detection probes including a fourth detection probe comprising the sequence of SEQ ID NO:25 and one or two detection probes selected from a group consisting of a fifth detection probe comprising the sequence of SEQ ID NO:26, a sixth detection probe comprising the sequence of SEQ ID NO:48 and a seventh detection probe consisting of the sequence of SEQ ID NO:49, and the gDNA solution by concurrently exposing the gDNA solution to the second set of detection probes, wherein:

the at least second nucleotide is located at a second particular position, independent of the position of the at least a nucleotide, of the TGFβI gene corresponding to a second single nucleotide polymorphism (SNP) associated with corneal dystrophy.