# Patent

## Title
Classification and diagnosis of the molecular basis of cholestasis

## Abstract

The methods and compositions of the invention find use in the clinical diagnosis of cholestasis related syndromes, particularly PFIC types 1, 2, and 3; BRIC types 1 and 2; Alagille syndrome, and alpha1-antitrypsin deficiency. The compositions of the invention include isolated nucleic acid molecules and oligonucleotide pairs suitable for use in amplifying regions of cholestasis related genes. Compositions of the invention include a cholestasis related gene resequencing microarray suitable for determining the nucleotide sequence of a region of a cholestasis related gene. Knowledge of the nucleotide sequence of one or more regions of a patient's cholestasis related gene allows diagnosis of the patient's syndrome.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of and claims priority to and benefit of PCT U.S. 2006/29372, filed on Jul. 28, 2006 which application claimed priority to U.S. Provisional Patent Application No. 60/703,703 filed on Jul. 29, 2005, the disclosures of which are incorporated herein by reference in their entirety.


FIELD OF THE INVENTION
The field of this invention relates to the field of genetic diagnostic assays for cholestasis related syndromes.
BACKGROUND OF THE INVENTION
Pathological jaundice (here used as a synonym for cholestasis) is the most frequent presenting syndrome in children and adults with liver disease. It results from the impaired excretion of bile from the liver, with a range of underlying causes including genetic, metabolic, inflammatory, and drug- or toxin-induced disorders. Therefore, physicians are challenged to identify the cause of jaundice in order to initiate specific treatment and supportive measures to prevent progression and/or complications of liver disease. The prognosis and treatment course for these cholestasis related syndromes differ widely. Thus, identifying the precise cause of cholestasis or jaundice allows appropriate treatment to be provided to a patient exhibiting cholestasis.
Progressive familial intrahepatic cholestasis (PFIC), benign recurring intrahepatic cholestasis (BRIC), alpha1-antitrypsin deficiency, and Alagille disease constitute approximately 45% of neonatal jaundice cases. These diseases can be difficult to clinically define. The number and size of the cholestasis related genes underlying PFIC, BRIC, alpha1-antitrypsin deficiency, and Alagille disease has impaired the use of mutation detection as a diagnostic criterion. Cholestasis related genes underlying PFIC, BRIC, alpha1-antitrypsin deficiency, and Alagille disease include, but are not limited to, ATP8B1 (SEQ ID NO:71), ABCB11 (SEQ ID NO:72), ABCB4 (SEQ ID NO:73), JAG1 (SEQ ID NO:74), and SERPINA1 (SEQ ID NO:75). ATP8B1 is located on the long arm of chromosome 18 at 18q21.31. ABCB11 is located on the long arm of chromosome 2 at 2q24. ABCB4 is located on the long arm of chromosome 7 at 7q21.1. JAG1 is located on the short arm of chromosome 20 at 20p12.1-p11.23. SERPINA1 is located on the long arm of chromosome 14 at 14q32.1.
Development of efficient, accurate, sensitive methods of detecting mutations in the genes associated with cholestasis, particularly PFIC, BRIC, alpha1-antitrypsin deficiency, and Alagille syndrome is desirable. It is of importance to develop a method of predicting disease outcome with respect to treatment course. It is of particular importance to develop a method of determining cholestasis classification that would identify subjects likely to benefit from early aggressive treatment.
SUMMARY OF THE INVENTION
Compositions and methods for diagnosing cholestasis related syndromes are provided. The inventions are based on identification of nucleotide sequences for amplifying and sequencing the 5′ regulatory regions, exons, and exon-intron boundaries of several cholestasis related genes: ATP8B1 (SEQ ID NO:71); ABCB11 (SEQ ID NO:72); ABCB4 (SEQ ID NO:73); JAG1 (SEQ ID NO:74); and SERPIN-1 (SEQ ID NO:75). The compositions of the invention allow amplification of the cholestasis related gene exons; exon-intron boundaries; and 5′ regulatory regions and sequencing of the cholestasis related gene exons, exon-intron boundaries, and the 5′ upstream region. The cholestasis related gene exon, exon-intron boundary, and the 5′ upstream region nucleotide sequences provide prognostic and diagnostic information for cholestasis related syndromes.
Compositions of the invention include isolated nucleic acid molecules comprising the nucleotide sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, and variants thereof. Variant nucleotide sequences of the invention differ by one nucleotide alteration from the nucleotide sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, or hybridize under stringent conditions to a complement of a nucleotide sequence of the invention.
Compositions of the invention include an oligonucleotide pair library comprised of at least one oligonucleotide pair. Compositions of the invention further include oligonucleotide pairs comprising a first nucleic acid molecule and a second nucleic acid molecule. Oligonucleotide pairs of the invention allow amplification of a region of a cholestasis related gene. Nucleotide sequences of the first nucleic acid molecule in an oligonucleotide pair are set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, and variants thereof. Nucleotide sequences of the second nucleic acid molecule in an oligonucleotide pair are set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and variants thereof.
In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:1 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:2 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:3 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:4 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:5 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:6 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:7 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:8 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:9 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:10 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:11 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:12 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:13 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:14 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:15 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:16 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:17 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:18 or a variant thereof. In an embodiment an oligonucleotide pair of the invention allows amplification of a region of the ATP8B1 protein gene (SEQ ID NO:71).
In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:19 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:20 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:21 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:22 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:23 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:24 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:25 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:26 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:27 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:28 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:29 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:30 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:31 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:32 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:33 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:34 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:35 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:36 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:37 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:38 or a variant thereof. In an embodiment an oligonucleotide pair of the invention allows amplification of a region of the ABCB11 protein gene (SEQ ID NO:72).
In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:39 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:40 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:41 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:42 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:43 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:44 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:45 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:46 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:47 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:48 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:49 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:50 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:51 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:52 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:53 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:54 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:55 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:56 or a variant thereof. In an embodiment an oligonucleotide pair of the invention allows amplification of a region of the ABCB4 protein gene (SEQ ID NO:73).
In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:57 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:58 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:59 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:60 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:61 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:62 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:63 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:64 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:65 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:66 or a variant thereof. In an embodiment an oligonucleotide pair of the invention allows amplification of a region of the JAG1 protein gene (SEQ ID NO:74).
In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:67 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:68 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:69 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:70 or a variant thereof. In an embodiment an oligonucleotide pair of the invention allows amplification of a region of the SERPINA-1 protein gene (SEQ ID NO:75).
Compositions of the invention include a resequencing microarray consisting of nucleic acid probes having a nucleotide sequence derived from the nucleotide sequences set forth in SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75. The nucleic acid probes are of a predetermined length, such as 25 nucleotides. Each probe has a predetermined nucleotide sequence that is a segment of the nucleotide sequence set forth in nucleotides 1 to 483 of SEQ ID NO:71; nucleotides 25,479 to 25,620 of SEQ ID NO:71; nucleotides 27,398 to 27,555 of SEQ ID NO:71; nucleotides 30,753 to 30,895 of SEQ ID NO:71; nucleotides 30,966 to 31,071 of SEQ ID NO:71; nucleotides 34,199 to 34,315 of SEQ ID NO:71; nucleotides 34,378 to 34,492 of SEQ ID NO:71; nucleotides 36,534 to 36,660 of SEQ ID NO:71; nucleotides 36,737 to 36,939 of SEQ ID NO:71; nucleotides 37,396 to 37,528 of SEQ ID NO:71; nucleotides 40,069 to 40,303 of SEQ ID NO:71; nucleotides 43,559 to 43,811 of SEQ ID NO:71; nucleotides 46,951 to 47,038 of SEQ ID NO:71; nucleotides 47,874 to 48,074 of SEQ ID NO:71; nucleotides 57,044 to 57,276 of SEQ ID NO:71; nucleotides 60,486 to 60,642 of SEQ ID NO:71; nucleotides 62,584 to 62,792 of SEQ ID NO:71; nucleotides 63,526 to 63,681 of SEQ ID NO:71; nucleotides 64,899 to 65,018 of SEQ ID NO:71; nucleotides 69,451 to 69,627 of SEQ ID NO:71; nucleotides 70,604 to 70,936 of SEQ ID NO:71; nucleotides 76,649 to 76,916 of SEQ ID NO:71; nucleotides 77,991 to 78,118 of SEQ ID NO:71; nucleotides 79,337 to 79,626 of SEQ ID NO:71; nucleotides 79,894 to 80,076 of SEQ ID NO:71; nucleotides 81,569 to 81,743 of SEQ ID NO:71; nucleotides 83,354 to 83,756 of SEQ ID NO:71; nucleotides 1 to 401 of SEQ ID NO:72; nucleotides 13,430 to 13,576 of SEQ ID NO:72; nucleotides 14,783 to 14,848 of SEQ ID NO:72; nucleotides 17,228 to 17,323 of SEQ ID NO:72; nucleotides 18,072 to 18,354 of SEQ ID NO:72; nucleotides 34,860 to 34,991 of SEQ ID NO:72; nucleotides 36,100 to 36,277 of SEQ ID NO:72; nucleotides 37,700 to 37,915 of SEQ ID NO:72; nucleotides 40,657 to 40,825 of SEQ ID NO:72; nucleotides 45,298 to 45,516 of SEQ ID NO:72; nucleotides 51,603 to 51,760 of SEQ ID NO:72; nucleotides 54,895 to 55,049 of SEQ ID NO:72; nucleotides 57,742 to 57,911 of SEQ ID NO:72; nucleotides 59,532 to 59,779 of SEQ ID NO:72; nucleotides 61,367 to 61,581 of SEQ ID NO:72; nucleotides 62,031 to 62,276 of SEQ ID NO:72; nucleotides 63,092 to 63,199 of SEQ ID NO:72; nucleotides 67,274 to 67,420 of SEQ ID NO:72; nucleotides 73,454 to 73,662 of SEQ ID NO:72; nucleotides 86,621 to 86,769 of SEQ ID NO:72; nucleotides 86,816 to 87,021 of SEQ ID NO:72; nucleotides 95,149 to 95,396 of SEQ ID NO:72; nucleotides 96,157 to 96,442 of SEQ ID NO:72; nucleotides 99,049 to 99,249 of SEQ ID NO:72; nucleotides 100,720 to 100,961 of SEQ ID NO:72; nucleotides 104,220 to 104,470 of SEQ ID NO:72; nucleotides 106,779 to 106,969 of SEQ ID NO:72; nucleotides 107,760 to 108,687 of SEQ ID NO:72; nucleotides 1 to 1,802 of SEQ ID NO:73; nucleotides 1,803 to 2,186 of SEQ ID NO:73; nucleotides 2,299 to 2,400 of SEQ ID NO:73; nucleotides 3,075 to 3,254 of SEQ ID NO:73; nucleotides 3,611 to 3,760 of SEQ ID NO:73; nucleotides 3,879 to 4,008 of SEQ ID NO:73; nucleotides 6,675 to 6,773 of SEQ ID NO:73; nucleotides 16,442 to 16,636 of SEQ ID NO:73; nucleotides 24,758 to 24,859 of SEQ ID NO:73; nucleotides 26,215 to 26,450 of SEQ ID NO:73; nucleotides 27,556 to 27,771 of SEQ ID NO:73; nucleotides 29,258 to 29,426 of SEQ ID NO:73; nucleotides 32,145 to 32,360 of SEQ ID NO:73; nucleotides 34,375 to 34,532 of SEQ ID NO:73; nucleotides 35,577 to 35,731 of SEQ ID NO:73; nucleotides 35,906 to 36,075 of SEQ ID NO:73; nucleotides 38,948 to 39,195 of SEQ ID NO:73; nucleotides 39,513 to 39,721 727 of SEQ ID NO:73; nucleotides 47,785 to 47,990 of SEQ ID NO:73; nucleotides 52,430 to 52,644 of SEQ ID NO:73; nucleotides 55,298 to 55,488 of SEQ ID NO:73; nucleotides 57,125 to 57,273 of SEQ ID NO:73; nucleotides 59,275 to 59,396 of SEQ ID NO:73; nucleotides 60,730 to 60,857 of SEQ ID NO:73; nucleotides 61,835 to 62,082 of SEQ ID NO:73; nucleotides 65,633 to 65,777 of SEQ ID NO:73; nucleotides 67,317 to 67,501 of SEQ ID NO:73; nucleotides 69,958 to 70,158 of SEQ ID NO:73; nucleotides 71,116 to 71,357 of SEQ ID NO:73; nucleotides 72,835 to 73,106 of SEQ ID NO:73; nucleotides 76,069 to 76,259 of SEQ ID NO:73; nucleotides 77,048 to 77,349 of SEQ ID NO:73; nucleotides 1 to 1,015 of SEQ ID NO:74; nucleotides 1,415 to 1,764 of SEQ ID NO:74; nucleotides 6,490 to 6,689 of SEQ ID NO:74; nucleotides 8,080 to 8,239 of SEQ ID NO:74; nucleotides 10,407 to 10,502 of SEQ ID NO:74; nucleotides 15,699 to 15,997 of SEQ ID NO:74; nucleotides 17,963 to 18,067 of SEQ ID NO:74; nucleotides 21,823 to 21,997 of SEQ ID NO:74; nucleotides 22,171 to 22,334 of SEQ ID NO:74; nucleotides 22,727 to 22,884 of SEQ ID NO:74; nucleotides 24,061 to 24,218 of SEQ ID NO:74; nucleotides 24,786 to 24,943 of SEQ ID NO:74; nucleotides 25,314 to 25,404 of SEQ ID NO:74; nucleotides 25,699 to 25,916 of SEQ ID NO:74; nucleotides 26,311 to 26,505 of SEQ ID NO:74; nucleotides 27,318 to 27,526 of SEQ ID NO:74; nucleotides 28,337 to 28,494 of SEQ ID NO:74; nucleotides 28,952 to 29,109 of SEQ ID NO:74; nucleotides 29,165 to 29,322 of SEQ ID NO:74; nucleotides 29,442 to 29,602 of SEQ ID NO:74; nucleotides 30,037 to 30,108 of SEQ ID NO:74; nucleotides 30,558 to 30,687 of SEQ ID NO:74; nucleotides 31,820 to 31,977 of SEQ ID NO:74; nucleotides 32,529 to 32,682 of SEQ ID NO:74; nucleotides 32,728 to 33,005 of SEQ ID NO:74; nucleotides 33,177 to 33,352 of SEQ ID NO:74; nucleotides 33,488 to 33,682 of SEQ ID NO:74; nucleotides 34,466 to 36,779 of SEQ ID NO:74; nucleotides 1 to 538 of SEQ ID NO:75; nucleotides 539 to 792 of SEQ ID NO:75; nucleotides 886 to 1,033 of SEQ ID NO:75; nucleotides 6,308 to 7,001 of SEQ ID NO:75; nucleotides 8,408 to 8,722 of SEQ ID NO:75; nucleotides 9,938 to 10,129 of SEQ ID NO:75; or nucleotides 10,909 to 11,232 of SEQ ID NO:75 or differs by one predetermined nucleotide alteration from a segment of the nucleotide sequence set forth in nucleotides 1 to 483 of SEQ ID NO:71; nucleotides 25,479 to 25,620 of SEQ ID NO:71; nucleotides 27,398 to 27,555 of SEQ ID NO:71; nucleotides 30,753 to 30,895 of SEQ ID NO:71; nucleotides 30,966 to 31,071 of SEQ ID NO:71; nucleotides 34,199 to 34,315 of SEQ ID NO:71; nucleotides 34,378 to 34,492 of SEQ ID NO:71; nucleotides 36,534 to 36,660 of SEQ ID NO:71; nucleotides 36,737 to 36,939 of SEQ ID NO:71; nucleotides 37,396 to 37,528 of SEQ ID NO:71; nucleotides 40,069 to 40,303 of SEQ ID NO:71; nucleotides 43,559 to 43,811 of SEQ ID NO:71; nucleotides 46,951 to 47,038 of SEQ ID NO:71; nucleotides 47,874 to 48,074 of SEQ ID NO:71; nucleotides 57,044 to 57,276 of SEQ ID NO:71; nucleotides 60,486 to 60,642 of SEQ ID NO:71; nucleotides 62,584 to 62,792 of SEQ ID NO:71; nucleotides 63,526 to 63,681 of SEQ ID NO:71; nucleotides 64,899 to 65,018 of SEQ ID NO:71; nucleotides 69,451 to 69,627 of SEQ ID NO:71; nucleotides 70,604 to 70,936 of SEQ ID NO:71; nucleotides 76,649 to 76,916 of SEQ ID NO:71; nucleotides 77,991 to 78,118 of SEQ ID NO:71; nucleotides 79,337 to 79,626 of SEQ ID NO:71; nucleotides 79,894 to 80,076 of SEQ ID NO:71; nucleotides 81,569 to 81,743 of SEQ ID NO:71; nucleotides 83,354 to 83,756 of SEQ ID NO:71; nucleotides 1 to 401 of SEQ ID NO:72; nucleotides 13,430 to 13,576 of SEQ ID NO:72; nucleotides 14,783 to 14,848 of SEQ ID NO:72; nucleotides 17,228 to 17,323 of SEQ ID NO:72; nucleotides 18,072 to 18,354 of SEQ ID NO:72; nucleotides 34,860 to 34,991 of SEQ ID NO:72; nucleotides 36,100 to 36,277 of SEQ ID NO:72; nucleotides 37,700 to 37,915 of SEQ ID NO:72; nucleotides 40,657 to 40,825 of SEQ ID NO:72; nucleotides 45,298 to 45,516 of SEQ ID NO:72; nucleotides 51,603 to 51,760 of SEQ ID NO:72; nucleotides 54,895 to 55,049 of SEQ ID NO:72; nucleotides 57,742 to 57,911 of SEQ ID NO:72; nucleotides 59,532 to 59,779 of SEQ ID NO:72; nucleotides 61,367 to 61,581 of SEQ ID NO:72; nucleotides 62,031 to 62,276 of SEQ ID NO:72; nucleotides 63,092 to 63,199 of SEQ ID NO:72; nucleotides 67,274 to 67,420 of SEQ ID NO:72; nucleotides 73,454 to 73,662 of SEQ ID NO:72; nucleotides 86,621 to 86,769 of SEQ ID NO:72; nucleotides 86,816 to 87,021 of SEQ ID NO:72; nucleotides 95,149 to 95,396 of SEQ ID NO:72; nucleotides 96,157 to 96,442 of SEQ ID NO:72; nucleotides 99,049 to 99,249 of SEQ ID NO:72; nucleotides 100,720 to 100,961 of SEQ ID NO:72; nucleotides 104,220 to 104,470 of SEQ ID NO:72; nucleotides 106,779 to 106,969 of SEQ ID NO:72; nucleotides 107,760 to 108,687 of SEQ ID NO:72; nucleotides 1 to 1,802 of SEQ ID NO:73; nucleotides 1,803 to 2,186 of SEQ ID NO:73; nucleotides 2,299 to 2,400 of SEQ ID NO:73; nucleotides 3,075 to 3,254 of SEQ ID NO:73; nucleotides 3,611 to 3,760 of SEQ ID NO:73; nucleotides 3,879 to 4,008 of SEQ ID NO:73; nucleotides 6,675 to 6,773 of SEQ ID NO:73; nucleotides 16,442 to 16,636 of SEQ ID NO:73; nucleotides 24,758 to 24,859 of SEQ ID NO:73; nucleotides 26,215 to 26,450 of SEQ ID NO:73; nucleotides 27,556 to 27,771 of SEQ ID NO:73; nucleotides 29,258 to 29,426 of SEQ ID NO:73; nucleotides 32,145 to 32,360 of SEQ ID NO:73; nucleotides 34,375 to 34,532 of SEQ ID NO:73; nucleotides 35,577 to 35,731 of SEQ ID NO:73; nucleotides 35,906 to 36,075 of SEQ ID NO:73; nucleotides 38,948 to 39,195 of SEQ ID NO:73; nucleotides 39,513 to 39,727 of SEQ ID NO:73; nucleotides 47,785 to 47,990 of SEQ ID NO:73; nucleotides 52,430 to 52,644 of SEQ ID NO:73; nucleotides 55,298 to 55,488 of SEQ ID NO:73; nucleotides 57,125 to 57,273 of SEQ ID NO:73; nucleotides 59,275 to 59,396 of SEQ ID NO:73; nucleotides 60,730 to 60,857 of SEQ ID NO:73; nucleotides 61,835 to 62,082 of SEQ ID NO:73; nucleotides 65,633 to 65,777 of SEQ ID NO:73; nucleotides 67,317 to 67,501 of SEQ ID NO:73; nucleotides 69,958 to 70,158 of SEQ ID NO:73; nucleotides 71,116 to 71,357 of SEQ ID NO:73; nucleotides 72,835 to 73,106 of SEQ ID NO:73; nucleotides 76,069 to 76,259 of SEQ ID NO:73; nucleotides 77,048 to 77,349 of SEQ ID NO:73; nucleotides 1 to 1,015 of SEQ ID NO:74; nucleotides 1,415 to 1,764 of SEQ ID NO:74; nucleotides 6,490 to 6,689 of SEQ ID NO:74; nucleotides 8,080 to 8,239 of SEQ ID NO:74; nucleotides 10,407 to 10,502 of SEQ ID NO:74; nucleotides 15,699 to 15,997 of SEQ ID NO:74; nucleotides 17,963 to 18,067 of SEQ ID NO:74; nucleotides 21,823 to 21,997 of SEQ ID NO:74; nucleotides 22,171 to 22,334 of SEQ ID NO:74; nucleotides 22,727 to 22,884 of SEQ ID NO:74; nucleotides 24,061 to 24,218 of SEQ ID NO:74; nucleotides 24,786 to 24,943 of SEQ ID NO:74; nucleotides 25,314 to 25,404 of SEQ ID NO:74; nucleotides 25,699 to 25,916 of SEQ ID NO:74; nucleotides 26,311 to 26,505 of SEQ ID NO:74; nucleotides 27,318 to 27,526 of SEQ ID NO:74; nucleotides 28,337 to 28,494 of SEQ ID NO:74; nucleotides 28,952 to 29,109 of SEQ ID NO:74; nucleotides 29,165 to 29,322 of SEQ ID NO:74; nucleotides 29,442 to 29,602 of SEQ ID NO:74; nucleotides 30,037 to 30,108 of SEQ ID NO:74; nucleotides 30,558 to 30,687 of SEQ ID NO:74; nucleotides 31,820 to 31,977 of SEQ ID NO:74; nucleotides 32,529 to 32,682 of SEQ ID NO:74; nucleotides 32,728 to 33,005 of SEQ ID NO:74; nucleotides 33,177 to 33,352 of SEQ ID NO:74; nucleotides 33,488 to 33,682 of SEQ ID NO:74; nucleotides 34,466 to 36,779 of SEQ ID NO:74; nucleotides 1 to 538 of SEQ ID NO:75; nucleotides 539 to 792 of SEQ ID NO:75; nucleotides 886 to 1,033 of SEQ ID NO:75; nucleotides 6,308 to 7,001 of SEQ ID NO:75; nucleotides 8,408 to 8,722 of SEQ ID NO:75; nucleotides 9,938 to 10,129 of SEQ ID NO:75; or nucleotides 10,909 to 11,232 of SEQ ID NO:75.
Compositions of the invention include a kit comprising a cholestasis related gene resequencing microarray and at least one oligonucleotide pair of the invention. In an aspect, the kit further comprises a standard sequence profile.
Methods of the invention include methods of determining the nucleotide sequence of a region of a cholestasis related gene of a human subject. The method involves the steps of obtaining a biological sample from the subject, providing an oligonucleotide pair of the invention, performing enzymatic amplification of a region of a cholestasis related gene, providing amplified DNA, and determining the nucleotide sequence of a region of a cholestasis related gene. In an aspect, the method involves the step of providing a cholestasis related gene resequencing microarray. In an aspect, the nucleotide sequence of one or multiple amplified regions is determined. In an aspect the nucleotide sequence of a region of at least one, two, three, four, or five cholestasis related genes is compared with a standard sequence profile. In an aspect of the invention, the cholestasis related gene is selected from the group consisting of ATP8B1 (SEQ ID NO:71), ABCB4 (SEQ ID NO:73), ABCB11 (SEQ ID NO:72), JAG1 (SEQ ID NO:74), and SERPINA-1 (SEQ ID NO:75).
Methods of the invention include methods of diagnosing a cholestasis related syndrome. The methods involve the steps of obtaining a biological sample from a human subject exhibiting cholestasis, performing enzymatic amplification of a region of a cholestasis related gene, providing amplified DNA having the nucleotide sequence of a region of a cholestasis related gene, determining the nucleotide sequence of the amplified DNA, and comparing the nucleotide sequence of the amplified DNA with a standard sequence profile. In an aspect, the method comprises the step of providing a cholestasis related gene resequencing microarray. Cholestasis related syndromes include, but are not limited to, cholestasis, neonatal cholestasis, progressive familial intrahepatic cholestasis, benign recurrent intrahepatic cholestasis, alpha-1 anti-trypsin deficiency, Alagille syndrome, intrahepatic cholestasis of pregnancy, primary sclerosing cholangitis, biliary atresia, cholelithiasis, cirrhosis of the liver, and septicemia. In an aspect, the method comprises the step of providing at least one oligonucleotide pair of the invention. In an aspect, enzymatic amplification of multiple regions, including regions of multiple cholestasis related genes, occurs. In an aspect, the nucleotide sequences of multiple amplified DNA are determined.
Methods of the invention include methods of determining the cholestasis classification of a subject exhibiting cholestasis. The methods comprise the steps of obtaining a biological sample from the subject, determining the nucleotide sequence of a region of a cholestasis related gene of the human subject, and comparing the nucleotide sequence of the region with a standard sequence profile. In an aspect of the method, the step of determining the nucleotide sequence further comprises the steps of performing enzymatic amplification of a region of a cholestasis related gene, providing amplified DNA having the nucleotide sequence of a region of a cholestasis related gene, and providing a cholestasis related gene resequencing microarray.
Methods of the invention include methods of predicting the prognosis for a subject exhibiting cholestasis. The methods comprise the steps of obtaining a biological sample from the subject, determining the nucleotide sequence of a region of a cholestasis related gene of the human subject, and comparing the nucleotide sequence of the region with a standard sequence profile. In an aspect of the method, the step of determining the nucleotide sequence further comprises the steps of performing enzymatic amplification of a region of a cholestasis related gene, providing amplified DNA having the nucleotide sequence of a region of a cholestasis related gene, and providing a cholestasis related gene resequencing microarray.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 presents the ATP8B1 oligonucleotide primer pairs, the amplicon size, the relative genomic location of the amplicon, and the size of the targets tiled on the resequencing microarray in Panel A. Panel B presents working oligonucleotide names, the nucleotide sequence of each primer, and the SEQ ID NO. Panel C presents a photograph of the PCR amplification products. PCR reactions were performed with the indicated oligonucleotide pair: Lane 1 (SEQ ID NO:1 and SEQ ID NO:2), Lane 2 (SEQ ID NO:3 and SEQ ID NO:4), Lane 3 (SEQ ID NO:5 and SEQ ID NO:6), Lane 4 (SEQ ID NO:7 and SEQ ID NO:8), Lane 5 (SEQ ID NO:9 and SEQ ID NO:10), Lane 6 (SEQ ID NO:11 and SEQ ID NO:12), Lane 7 (SEQ ID NO:13 and SEQ ID NO:14), Lane 8 (SEQ ID NO:15 and SEQ ID NO:16), Lane 9 (SEQ ID NO:17 and SEQ ID NO:18), and size markers (MW).
FIG. 2 presents the ABCB11 oligonucleotide primer pairs, the amplicon size, the relative genomic location of the amplicon, and the size of the targets tiled on the resequencing microarray in Panel A. Panel B presents working oligonucleotide names, the nucleotide sequence of each primer, and the SEQ ID NO. Panel C presents a photograph of the PCR amplification products. PCR reactions were performed with the indicated oligonucleotide pair: Lane 1 (SEQ ID NO:19 and SEQ ID NO:20), Lane 2 (SEQ ID NO:21 and SEQ ID NO:22), Lane 3 (SEQ ID NO:23 and SEQ ID NO:24), Lane 4 (SEQ ID NO:25 and SEQ ID NO:26), Lane 5 (SEQ ID NO:27 and SEQ ID NO:28), Lane 6 (SEQ ID NO:29 and SEQ ID NO:30), Lane 7 (SEQ ID NO:31 and SEQ ID NO:32), Lane 8 (SEQ ID NO:33 and SEQ ID NO:34), Lane 9 (SEQ ID NO:35 and SEQ ID NO:36), Lane 10 (SEQ ID NO:37 and SEQ ID NO:38), and size markers (MW).
FIG. 3 presents the ABCB4 oligonucleotide primer pairs, the amplicon size, the relative genomic location of the amplicon, and the size of the targets tiled on the resequencing microarray in Panel A. Panel B presents working oligonucleotide names, the nucleotide sequence of each primer, and the SEQ ID NO. Panel C presents a photograph of the PCR amplification products. PCR reactions were performed with the indicated oligonucleotide pair: Lane 1 (SEQ ID NO:39 and SEQ ID NO:40), Lane 2 (SEQ ID NO:41 and SEQ ID NO:42), Lane 3 (SEQ ID NO:43 and SEQ ID NO:44), Lane 4 (SEQ ID NO:45 and SEQ ID NO:46), Lane 5 (SEQ ID NO:47 and SEQ ID NO:48), Lane 6 (SEQ ID NO:49 and SEQ ID NO:50), Lane 7 (SEQ ID NO:51 and SEQ ID NO:52), Lane 8 (SEQ ID NO:53 and SEQ ID NO:54), Lane 9 (SEQ ID NO:55 and SEQ ID NO:56), and size markers (MW).
FIG. 4 presents the JAG1 oligonucleotide primer pairs, the amplicon size, the relative genomic location of the amplicon, and the size of the targets tiled on the resequencing microarray in Panel A. Panel B presents working oligonucleotide names, the nucleotide sequence of each primer, and the SEQ ID NO. Panel C presents a photograph of the PCR amplification products. PCR reactions were performed with the indicated oligonucleotide pair: Lane 1 (SEQ ID NO:57 and SEQ ID NO:58), Lane 2 (SEQ ID NO:59 and SEQ ID NO:60), Lane 3 (SEQ ID NO:61 and SEQ ID NO:62), Lane 4 (SEQ ID NO:63 and SEQ ID NO:64), Lane 5 (SEQ ID NO:65 and SEQ ID NO:66), and size markers (MW).
FIG. 5 presents the SERPINA 1 oligonucleotide primer pairs, the amplicon size, the relative genomic location of the amplicon, and the size of the targets tiled on the resequencing microarray in Panel A. Panel B presents working oligonucleotide names, the nucleotide sequence of each primer, and the SEQ ID NO. Panel C presents a photograph of the PCR amplification products. PCR reactions were performed with the indicated oligonucleotide pair: Lane 1 (SEQ ID NO:67 and SEQ ID NO:68), Lane 2 (SEQ ID NO:69 and SEQ ID NO:70), and size markers (MW).



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods of determining the nucleotide sequence of a region of a cholestasis related gene, classifying disease, particularly cholestasis related syndromes, diagnosing a cholestasis related syndrome, and predicting the prognosis of a cholestasis related syndrome. The invention provides methods of amplifying a region or regions of various cholestasis related genes, and methods of determining the nucleotide sequence of a region or regions of various cholestasis related genes. Compositions of the invention include isolated nucleic acid molecules and oligonucleotide pairs useful in amplifying a region or regions of various cholestasis related genes as well as kits for performing the methods of the invention. Compositions of the invention include a resequencing microarray useful in determining the nucleotide sequence of the promoter region, exon regions, and exon/intron boundaries of various cholestasis related genes as well as kits for performing the methods of the invention. The invention relates to identification of genomic nucleotide alterations in a cholestasis related gene in various cholestasis related syndromes.
Cholestasis related syndromes are characterized by an impaired excretion of bile from the liver resulting in elevated bilirubin levels in the bloodstream. Cholestasis related syndromes include, but are not limited to, pathological jaundice, cancer, obstruction of the biliary duct by gall stones, inflammatory masses, or tumors, cholecystitis, pancreatic carcinoma, cholestasis, Gilbert's disease, acetaminophen overdose, hepatitis B infection, hemolysis, hereditary spherocytosis, conjugated hyperbilirubinemia, acholuric jaundice, breastfeeding jaundice, breast milk jaundice, cholestatic jaundice, congenital jaundice, hematogenous jaundice, hemolytic jaundice, hemorrhagic jaundice, hepatocellular jaundice, infectious jaundice, jaundice of the newborn, nonhemolytic jaundice, obstructive jaundice, pathological jaundice of the newborn, parenchymatous jaundice, retention jaundice, Alagille disease, biliary atresia, progressive familial intrahepatic cholestasis (PFIC), PFIC type 1, PFIC type II, and PFIC type III, benign recurring intrahepatic cholestasis (BRIC), BRIC type I, BRIC type II, alpha1-antitrypsin deficiency, Alagille syndrome, intrahepatic cholestasis of pregnancy, primary sclerosing cholangitis, neonatal cholestasis, cholelithiasis, cirrhosis of the liver, cholangitis, Byler Disease, Byler Syndrome, cholelithiasis, liver disease related to total parenteral nutrition, chronic lung disease, congenital heart disease, exocrine pancreatic deficiency, and septicemia.
Cholestasis related phenotypes include, but are not limited to, jaundice, altered enzyme levels, growth failure, discolored urine, discolored stools, pruritis, abnormal bleeding, steatorrhea, elevated alkaline phosphatase levels, altered bilirubin levels, clotting rate, fatigue, loss of appetite, asthma-like symptoms, hyperlipidemia, xanthoma formation, and cirrhosis.
Methods of measuring cholestasis related phenotypes include, but are not limited to, physical examination, bilirubin assays, ultrasounds, computed tomography, liver biopsy, endoscopy, magnetic resonance imaging, enzymatic assays including, but not limited to, alanine transaminase, albumin, alkaline phosphatase, alpha-fetoprotein, aspartate transaminase, gamma-glutamyl transpeptidase, lactic dehydrogenase, mitochondrial antibodies, 5′-nucleotidase, and prothrombin assays; endoscopic retrograde cholangiopancreatography, percutaneous transhepatic cholangiography, and operative cholangiography.
Compositions of the invention include isolated nucleic acid molecules having the nucleotide sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70, or a fragment or variants thereof. Isolated nucleic acid molecules of the invention anneal to a nucleotide sequence of interest.
Nucleotide sequences of interest in the present invention include numerous cholestasis related compounds. Cholestasis related genes include, but are not limited to, ATP8B1 (SEQ ID NO:71); ABCB11 (SEQ ID NO:72); ABCB4 (SEQ ID NO:73); JAG1 (SEQ ID NO:74); and SERPINA-1 or SERPIN-1 (SEQ ID NO:75). Portions of the first intron sequence of ABCB4 (SEQ ID NO:73) and SERPINA-1 (SEQ ID NO:75) are not included in the sequence listing. The invention particularly pertains to nucleotide sequence alterations in the 5′ upstream regions, exons, and exon-intron boundary regions of the cholestasis related genes, thus selected regions of ATP8B1 (SEQ ID NO:71); ABCB11 (SEQ ID NO:72); ABCB4 (SEQ ID NO:73); JAG1 (SEQ ID NO:74); and SERPINA-1 (SEQ ID NO:75) are of particular interest.
Nucleic acid molecules having a nucleotide sequence set forth in SEQ ID NOS:1-18 anneal to the ATP8B1 gene (SEQ ID NO:71). Nucleic acid molecules having a nucleotide sequence set forth in SEQ ID NOS:19-38 anneal to the ABCB11 gene (SEQ ID NO:72). Nucleic acid molecules having a nucleotide sequence set forth in SEQ ID NOS:39-56 anneal to the ABCB4 gene (SEQ ID NO:73). Nucleic acid molecules having a nucleotide sequence set forth in SEQ ID NOS:57-66 anneal to the JAG1 gene (SEQ ID NO:74). Nucleic acid molecules having a nucleotide sequence set forth in SEQ ID NOS:67-70 anneal to the SERPINA-1 gene (SEQ ID NO:75).
The invention encompasses isolated or substantially purified nucleic acid compositions. An “isolated” or substantially “purified” nucleic acid molecule is substantially free of other cellular material, or culture medium when produced by recombinant techniques or substantially free of chemical precursors or other chemicals when chemically synthesized. A substantially purified preparation of nucleic acid molecules, particularly isolated nucleic acid molecules as used herein, means a preparation of nucleic acid molecules in which at least 70% of the nucleic acid molecules, preferably at least 80% of the nucleic acid molecules, more preferably at least 85% of the nucleic acid molecules, yet more preferably 90%, most preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the nucleic acid molecules are the subject sequence.
By fragments or variants thereof is intended isolated nucleic acid molecules having a nucleotide sequence that differs by one nucleotide alteration from that set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70 or a nucleotide sequence that hybridizes under stringent conditions to a complement of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70. A fragment or variant that differs by one nucleotide alteration from a nucleotide sequence set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 differs from that nucleotide sequence by the addition, insertion, deletion, removal, subtraction, or substitution of one nucleotide. Fragments or variants include isolated nucleic acid molecules having a nucleotide sequence that hybridizes under stringent conditions to a complement of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70.
By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a molecule will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Microarray hybridization conditions are discussed elsewhere herein. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a hybridization fragment is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.
By “region” is intended a portion of the entire nucleotide sequence of interest, such as the genomic ATP8B1 gene, ABCB11 gene, ABCB4 gene, JAG1 gene, and SERPINA-1 gene. A region of a nucleotide sequence of interest, such as a genomic cholestasis related gene may range from 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 4000, 4050, 4100, 4150, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 108687 nucleotides, or up to the total number of nucleotides present in a full-length nucleotide sequence of interest disclosed herein. Such a region contains nucleotide sequence from the 5′ regulatory control sequence, one or more exons, one or more exon-intron boundaries, or one or more introns of a cholestasis related nucleotide sequence of interest. The oligonucleotide pairs of the invention each amplify a different region of a cholestasis related gene. A region may comprise multiple smaller regions. For example, oligonucleotide pair of the invention may amplify a region comprising multiple regions of particular interest.
Regions of particular interest include, but are not limited to, the nucleotide sequences set forth in nucleotides 1 to 483 of SEQ ID NO:71; nucleotides 25,479 to 25,620 of SEQ ID NO:71; nucleotides 27,398 to 27,555 of SEQ ID NO:71; nucleotides 30,753 to 30,895 of SEQ ID NO:71; nucleotides 30,966 to 31,071 of SEQ ID NO:71; nucleotides 34,199 to 34,315 of SEQ ID NO:71; nucleotides 34,378 to 34,492 of SEQ ID NO:71; nucleotides 36,534 to 36,660 of SEQ ID NO:71; nucleotides 36,737 to 36,939 of SEQ ID NO:71; nucleotides 37,396 to 37,528 of SEQ ID NO:71; nucleotides 40,069 to 40,303 of SEQ ID NO:71; nucleotides 43,559 to 43,811 of SEQ ID NO:71; nucleotides 46,951 to 47,038 of SEQ ID NO:71; nucleotides 47,874 to 48,074 of SEQ ID NO:71; nucleotides 57,044 to 57,276 of SEQ ID NO:71; nucleotides 60,486 to 60,642 of SEQ ID NO:71; nucleotides 62,584 to 62,792 of SEQ ID NO:71; nucleotides 63,526 to 63,681 of SEQ ID NO:71; nucleotides 64,899 to 65,018 of SEQ ID NO:71; nucleotides 69,451 to 69,627 of SEQ ID NO:71; nucleotides 70,604 to 70,936 of SEQ ID NO:71; nucleotides 76,649 to 76,916 of SEQ ID NO:71; nucleotides 77,991 to 78,118 of SEQ ID NO:71; nucleotides 79,337 to 79,626 of SEQ ID NO:71; nucleotides 79,894 to 80,076 of SEQ ID NO:71; nucleotides 81,569 to 81,743 of SEQ ID NO:71; nucleotides 83,354 to 83,756 of SEQ ID NO:71; nucleotides 1 to 401 of SEQ ID NO:72; nucleotides 13,430 to 13,576 of SEQ ID NO:72; nucleotides 14,783 to 14,848 of SEQ ID NO:72; nucleotides 17,228 to 17,323 of SEQ ID NO:72; nucleotides 18,072 to 18,354 of SEQ ID NO:72; nucleotides 34,860 to 34,991 of SEQ ID NO:72; nucleotides 36,100 to 36,277 of SEQ ID NO:72; nucleotides 37,700 to 37,915 of SEQ ID NO:72; nucleotides 40,657 to 40,825 of SEQ ID NO:72; nucleotides 45,298 to 45,516 of SEQ ID NO:72; nucleotides 51,603 to 51,760 of SEQ ID NO:72; nucleotides 54,895 to 55,049 of SEQ ID NO:72; nucleotides 57,742 to 57,911 of SEQ ID NO:72; nucleotides 59,532 to 59,779 of SEQ ID NO:72; nucleotides 61,367 to 61,581 of SEQ ID NO:72; nucleotides 62,031 to 62,276 of SEQ ID NO:72; nucleotides 63,092 to 63,199 of SEQ ID NO:72; nucleotides 67,274 to 67,420 of SEQ ID NO:72; nucleotides 73,454 to 73,662 of SEQ ID NO:72; nucleotides 86,621 to 86,769 of SEQ ID NO:72; nucleotides 86,816 to 87,021 of SEQ ID NO:72; nucleotides 95,149 to 95,396 of SEQ ID NO:72; nucleotides 96,157 to 96,442 of SEQ ID NO:72; nucleotides 99,049 to 99,249 of SEQ ID NO:72; nucleotides 100,720 to 100,961 of SEQ ID NO:72; nucleotides 104,220 to 104,470 of SEQ ID NO:72; nucleotides 106,779 to 106,969 of SEQ ID NO:72; nucleotides 107,760 to 108,687 of SEQ ID NO:72; nucleotides 1 to 1,802 of SEQ ID NO:73; nucleotides 1,803 to 2,186 of SEQ ID NO:73; nucleotides 2,299 to 2,400 of SEQ ID NO:73; nucleotides 3,075 to 3,254 of SEQ ID NO:73; nucleotides 3,611 to 3,760 of SEQ ID NO:73; nucleotides 3,879 to 4,008 of SEQ ID NO:73; nucleotides 6,675 to 6,773 of SEQ ID NO:73; nucleotides 16,442 to 16,636 of SEQ ID NO:73; nucleotides 24,758 to 24,859 of SEQ ID NO:73; nucleotides 26,215 to 26,450 of SEQ ID NO:73; nucleotides 27,556 to 27,771 of SEQ ID NO:73; nucleotides 29,258 to 29,426 of SEQ ID NO:73; nucleotides 32,145 to 32,360 of SEQ ID NO:73; nucleotides 34,375 to 34,532 of SEQ ID NO:73; nucleotides 35,577 to 35,731 of SEQ ID NO:73; nucleotides 35,906 to 36,075 of SEQ ID NO:73; nucleotides 38,948 to 39,195 of SEQ ID NO:73; nucleotides 39,513 to 39,727 of SEQ ID NO:73; nucleotides 47,785 to 47,990 of SEQ ID NO:73; nucleotides 52,430 to 52,644 of SEQ ID NO:73; nucleotides 55,298 to 55,488 of SEQ ID NO:73; nucleotides 57,125 to 57,273 of SEQ ID NO:73; nucleotides 59,275 to 59,396 of SEQ ID NO:73; nucleotides 60,730 to 60,857 of SEQ ID NO:73; nucleotides 61,835 to 62,082 of SEQ ID NO:73; nucleotides 65,633 to 65,777 of SEQ ID NO:73; nucleotides 67,317 to 67,501 of SEQ ID NO:73; nucleotides 69,958 to 70,158 of SEQ ID NO:73; nucleotides 71,116 to 71,357 of SEQ ID NO:73; nucleotides 72,835 to 73,106 of SEQ ID NO:73; nucleotides 76,069 to 76,259 of SEQ ID NO:73; nucleotides 77,048 to 77,349 of SEQ ID NO:73; nucleotides 1 to 1,015 of SEQ ID NO:74; nucleotides 1,415 to 1,764 of SEQ ID NO:74; nucleotides 6,490 to 6,689 of SEQ ID NO:74; nucleotides 8,080 to 8,239 of SEQ ID NO:74; nucleotides 10,407 to 10,502 of SEQ ID NO:74; nucleotides 15,699 to 15,997 of SEQ ID NO:74; nucleotides 17,963 to 18,067 of SEQ ID NO:74; nucleotides 21,823 to 21,997 of SEQ ID NO:74; nucleotides 22,171 to 22,334 of SEQ ID NO:74; nucleotides 22,727 to 22,884 of SEQ ID NO:74; nucleotides 24,061 to 24,218 of SEQ ID NO:74; nucleotides 24,786 to 24,943 of SEQ ID NO:74; nucleotides 25,314 to 25,404 of SEQ ID NO:74; nucleotides 25,699 to 25,916 of SEQ ID NO:74; nucleotides 26,311 to 26,505 of SEQ ID NO:74; nucleotides 27,318 to 27,526 of SEQ ID NO:74; nucleotides 28,337 to 28,494 of SEQ ID NO:74; nucleotides 28,952 to 29,109 of SEQ ID NO:74; nucleotides 29,165 to 29,322 of SEQ ID NO:74; nucleotides 29,442 to 29,602 of SEQ ID NO:74; nucleotides 30,037 to 30,108 of SEQ ID NO:74; nucleotides 30,558 to 30,687 of SEQ ID NO:74; nucleotides 31,820 to 31,977 of SEQ ID NO:74; nucleotides 32,529 to 32,682 of SEQ ID NO:74; nucleotides 32,728 to 33,005 of SEQ ID NO:74; nucleotides 33,177 to 33,352 of SEQ ID NO:74; nucleotides 33,488 to 33,682 of SEQ ID NO:74; nucleotides 34,466 to 36,779 of SEQ ID NO:74; nucleotides 1 to 538 of SEQ ID NO:75; nucleotides 539 to 792 of SEQ ID NO:75; nucleotides 886 to 1,033 of SEQ ID NO:75; nucleotides 6,308 to 7,001 of SEQ ID NO:75; nucleotides 8,408 to 8,722 of SEQ ID NO:75; nucleotides 9,938 to 10,129 of SEQ ID NO:75; or nucleotides 10,909 to 11,232 of SEQ ID NO:75.
Compositions of the invention include oligonucleotide pairs. An oligonucleotide pair of the invention consists of a first isolated nucleic acid molecule of the invention and a second isolated nucleic acid molecule of the invention with different nucleotide sequences. An oligonucleotide pair of the invention is suitable for use as a primer pair or primer set in an enzymatic amplification reaction such as, but not limited to, a PCR reaction. In an embodiment, an oligonucleotide pair comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, or a fragment or variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, or a fragment or variant thereof. The first and second nucleic acid molecules of an oligonucleotide pair anneal to opposite strands of a cholestasis related gene such that they allow amplification of a region of a cholestasis related gene bracketed by the primer pair.
In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:1 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:2 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:3 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:4 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:5 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:6 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:7 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:8 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:9 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:10 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:11 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:12 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:13 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:14 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:15 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:16 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:17 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:18 or a variant thereof. In an embodiment an oligonucleotide pair of the invention allows amplification of a region of the ATP8B1 protein gene (SEQ ID NO:71).
In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:19 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:20 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:21 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:22 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:23 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:24 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:25 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:26 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:27 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:28 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:29 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:30 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:31 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:32 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:33 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:34 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:35 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:36 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:37 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:38 or a variant thereof. In an embodiment an oligonucleotide pair of the invention allows amplification of a region of the ABCB11 protein gene (SEQ ID NO:72).
In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:39 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:40 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:41 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:42 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:43 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:44 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:45 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:46 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:47 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:48 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:49 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:50 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:51 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:52 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:53 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:54 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:55 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:56 or a variant thereof. In an embodiment an oligonucleotide pair of the invention allows amplification of a region of the ABCB4 protein gene (SEQ ID NO:73).
In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:57 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:58 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:59 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:60 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:61 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:62 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:63 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:64 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:65 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:66 or a variant thereof. In an embodiment an oligonucleotide pair of the invention allows amplification of a region of the JAG1 protein gene (SEQ ID NO:74).
In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:67 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:68 or a variant thereof. In an embodiment, an oligonucleotide pair of the invention comprises a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:69 or a variant thereof and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:70 or a variant thereof. In an embodiment an oligonucleotide pair of the invention allows amplification of a region of the SERPINA-1 protein gene (SEQ ID NO:75).
Additional oligonucleotide pairs comprising the isolated nucleic acid molecules of the invention are encompassed by the invention.
By “amplification” is intended an increase in the amount of nucleic acid molecules in a sample. Amplifying DNA increases the amount of acid precipitable nucleic acid molecules. The amount of acid precipitable material increases by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, or more. Methods of quantifying nucleic acid molecules are known in the art and include, but are not limited to, UV absorption spectra, radiolabel incorporation, agarose gel electrophoresis, bisbenzimide staining and ethidium bromide staining. See, for example, Ausubel et al., eds. (2003) Current Protocols in Molecular Biology, (John Wiley & Sons, New York) and Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y., herein incorporated by reference.
Enzymatic amplification is the process of using enzymes to perform amplification. A common type of enzymatic amplification is the polymerase chain reaction (PCR). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like. Known methods of PCR include, but are not limited to, methods using DNA polymerases from extremophiles, engineered DNA polymerases, and long-range PCR. It is recognized that it is preferable to use high fidelity PCR reaction conditions in the methods of the invention. See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Long range PCR amplification methods include methods such as those described in the TaKaRa LA PCR guide, Takara Shuzo Co., Ltd.
By “amplified DNA” is intended the product of enzymatic amplification.
Any method of DNA sequencing known in the art can be used in the methods of the invention. Methods of sequencing DNA are known in the art and are described in Graham & Hill eds. (2001) DNA Sequencing Protocols (Humana Press, Totowa N.J.), Kieleczawa ed (2004) DNA Sequencing: Optimizing the Process and Analysis (Jones & Bartlett Publishers, Ontario), Ausubel et al., eds. (2003) Current Protocols in Molecular Biology, (John Wiley & Sons, New York) and Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y., herein incorporated by reference in their entirety).
In an embodiment a resequencing microarray is used in the methods of the invention. By “resequencing microarray” or “variation detection array” is intended a microarray of nucleic acid probes arranged on a chip wherein each nucleic acid probe is of a predetermined length and sequence. Regions of a nucleotide sequence of interest are divided into segments of predetermined length and nucleic acid probes based on the sequence of each segment are arranged on a chip. Each nucleic acid probe contains a single query base. For each segment, four nucleic acid probes of identical sequence with the exception of the query base are tiled on the chip. A predetermined nucleotide is inserted at the query base location, and each nucleotide is presented in one of the four nucleic acid probes. Thus, each possible nucleotide sequence is represented. The sequence of each nucleic acid probe is identical to a segment of a nucleotide sequence of interest or differs by one predetermined nucleotide alteration from a segment of a nucleotide sequence of interest. See Warrington et al. (2002) Hum Mutat 19:402-409 and Cutler et al. (2001) Genome Res 11: 1913-1925, herein incorporated by reference in their entirety.
A cholestasis related gene resequencing microarray of the invention consists of nucleic acid probes having a nucleotide sequence derived from the nucleotide sequences set forth in SEQ ID NOS:71-75. In particular the nucleic acid probes have a nucleotide sequence derived from a segment of the nucleotide sequence set forth in nucleotides 1 to 483 of SEQ ID NO:71; nucleotides 25,479 to 25,620 of SEQ ID NO:71; nucleotides 27,398 to 27,555 of SEQ ID NO:71; nucleotides 30,753 to 30,895 of SEQ ID NO:71; nucleotides 30,966 to 31,071 of SEQ ID NO:71; nucleotides 34,199 to 34,315 of SEQ ID NO:71; nucleotides 34,378 to 34,492 of SEQ ID NO:71; nucleotides 36,534 to 36,660 of SEQ ID NO:71; nucleotides 36,737 to 36,939 of SEQ ID NO:71; nucleotides 37,396 to 37,528 of SEQ ID NO:71; nucleotides 40,069 to 40,303 of SEQ ID NO:71; nucleotides 43,559 to 43,811 of SEQ ID NO:71; nucleotides 46,951 to 47,038 of SEQ ID NO:71; nucleotides 47,874 to 48,074 of SEQ ID NO:71; nucleotides 57,044 to 57,276 of SEQ ID NO:71; nucleotides 60,486 to 60,642 of SEQ ID NO:71; nucleotides 62,584 to 62,792 of SEQ ID NO:71; nucleotides 63,526 to 63,681 of SEQ ID NO:71; nucleotides 64,899 to 65,018 of SEQ ID NO:71; nucleotides 69,451 to 69,627 of SEQ ID NO:71; nucleotides 70,604 to 70,936 of SEQ ID NO:71; nucleotides 76,649 to 76,916 of SEQ ID NO:71; nucleotides 77,991 to 78,118 of SEQ ID NO:71; nucleotides 79,337 to 79,626 of SEQ ID NO:71; nucleotides 79,894 to 80,076 of SEQ ID NO:71; nucleotides 81,569 to 81,743 of SEQ ID NO:71; nucleotides 83,354 to 83,756 of SEQ ID NO:71; nucleotides 1 to 401 of SEQ ID NO:72; nucleotides 13,430 to 13,576 of SEQ ID NO:72; nucleotides 14,783 to 14,848 of SEQ ID NO:72; nucleotides 17,228 to 17,323 of SEQ ID NO:72; nucleotides 18,072 to 18,354 of SEQ ID NO:72; nucleotides 34,860 to 34,991 of SEQ ID NO:72; nucleotides 36,100 to 36,277 of SEQ ID NO:72; nucleotides 37,700 to 37,915 of SEQ ID NO:72; nucleotides 40,657 to 40,825 of SEQ ID NO:72; nucleotides 45,298 to 45,516 of SEQ ID NO:72; nucleotides 51,603 to 51,760 of SEQ ID NO:72; nucleotides 54,895 to 55,049 of SEQ ID NO:72; nucleotides 57,742 to 57,911 of SEQ ID NO:72; nucleotides 59,532 to 59,779 of SEQ ID NO:72; nucleotides 61,367 to 61,581 of SEQ ID NO:72; nucleotides 62,031 to 62,276 of SEQ ID NO:72; nucleotides 63,092 to 63,199 of SEQ ID NO:72; nucleotides 67,274 to 67,420 of SEQ ID NO:72; nucleotides 73,454 to 73,662 of SEQ ID NO:72; nucleotides 86,621 to 86,769 of SEQ ID NO:72; nucleotides 86,816 to 87,021 of SEQ ID NO:72; nucleotides 95,149 to 95,396 of SEQ ID NO:72; nucleotides 96,157 to 96,442 of SEQ ID NO:72; nucleotides 99,049 to 99,249 of SEQ ID NO:72; nucleotides 100,720 to 100,961 of SEQ ID NO:72; nucleotides 104,220 to 104,470 of SEQ ID NO:72; nucleotides 106,779 to 106,969 of SEQ ID NO:72; nucleotides 107,760 to 108,687 of SEQ ID NO:72; nucleotides 1 to 1,802 of SEQ ID NO:73; nucleotides 1,803 to 2,186 of SEQ ID NO:73; nucleotides 2,299 to 2,400 of SEQ ID NO:73; nucleotides 3,075 to 3,254 of SEQ ID NO:73; nucleotides 3,611 to 3,760 of SEQ ID NO:73; nucleotides 3,879 to 4,008 of SEQ ID NO:73; nucleotides 6,675 to 6,773 of SEQ ID NO:73; nucleotides 16,442 to 16,636 of SEQ ID NO:73; nucleotides 24,758 to 24,859 of SEQ ID NO:73; nucleotides 26,215 to 26,450 of SEQ ID NO:73; nucleotides 27,556 to 27,771 of SEQ ID NO:73; nucleotides 29,258 to 29,426 of SEQ ID NO:73; nucleotides 32,145 to 32,360 of SEQ ID NO:73; nucleotides 34,375 to 34,532 of SEQ ID NO:73; nucleotides 35,577 to 35,731 of SEQ ID NO:73; nucleotides 35,906 to 36,075 of SEQ ID NO:73; nucleotides 38,948 to 39,195 of SEQ ID NO:73; nucleotides 39,513 to 39,727 of SEQ ID NO:73; nucleotides 47,785 to 47,990 of SEQ ID NO:73; nucleotides 52,430 to 52,644 of SEQ ID NO:73; nucleotides 55,298 to 55,488 of SEQ ID NO:73; nucleotides 57,125 to 57,273 of SEQ ID NO:73; nucleotides 59,275 to 59,396 of SEQ ID NO:73; nucleotides 60,730 to 60,857 of SEQ ID NO:73; nucleotides 61,835 to 62,082 of SEQ ID NO:73; nucleotides 65,633 to 65,777 of SEQ ID NO:73; nucleotides 67,317 to 67,501 of SEQ ID NO:73; nucleotides 69,958 to 70,158 of SEQ ID NO:73; nucleotides 71,116 to 71,357 of SEQ ID NO:73; nucleotides 72,835 to 73,106 of SEQ ID NO:73; nucleotides 76,069 to 76,259 of SEQ ID NO:73; nucleotides 77,048 to 77,349 of SEQ ID NO:73; nucleotides 1 to 1,015 of SEQ ID NO:74; nucleotides 1,415 to 1,764 of SEQ ID NO:74; nucleotides 6,490 to 6,689 of SEQ ID NO:74; nucleotides 8,080 to 8,239 of SEQ ID NO:74; nucleotides 10,407 to 10,502 of SEQ ID NO:74; nucleotides 15,699 to 15,997 of SEQ ID NO:74; nucleotides 17,963 to 18,067 of SEQ ID NO:74; nucleotides 21,823 to 21,997 of SEQ ID NO:74; nucleotides 22,171 to 22,334 of SEQ ID NO:74; nucleotides 22,727 to 22,884 of SEQ ID NO:74; nucleotides 24,061 to 24,218 of SEQ ID NO:74; nucleotides 24,786 to 24,943 of SEQ ID NO:74; nucleotides 25,314 to 25,404 of SEQ ID NO:74; nucleotides 25,699 to 25,916 of SEQ ID NO:74; nucleotides 26,311 to 26,505 of SEQ ID NO:74; nucleotides 27,318 to 27,526 of SEQ ID NO:74; nucleotides 28,337 to 28,494 of SEQ ID NO:74; nucleotides 28,952 to 29,109 of SEQ ID NO:74; nucleotides 29,165 to 29,322 of SEQ ID NO:74; nucleotides 29,442 to 29,602 of SEQ ID NO:74; nucleotides 30,037 to 30,108 of SEQ ID NO:74; nucleotides 30,558 to 30,687 of SEQ ID NO:74; nucleotides 31,820 to 31,977 of SEQ ID NO:74; nucleotides 32,529 to 32,682 of SEQ ID NO:74; nucleotides 32,728 to 33,005 of SEQ ID NO:74; nucleotides 33,177 to 33,352 of SEQ ID NO:74; nucleotides 33,488 to 33,682 of SEQ ID NO:74; nucleotides 34,466 to 36,779 of SEQ ID NO:74; nucleotides 1 to 538 of SEQ ID NO:75; nucleotides 539 to 792 of SEQ ID NO:75; nucleotides 886 to 1,033 of SEQ ID NO:75; nucleotides 6,308 to 7,001 of SEQ ID NO:75; nucleotides 8,408 to 8,722 of SEQ ID NO:75; nucleotides 9,938 to 10,129 of SEQ ID NO:75; or nucleotides 10,909 to 11,232 of SEQ ID NO:75 or differs by one predetermined nucleotide alteration from a segment of the nucleotide sequence set forth in nucleotides 1 to 483 of SEQ ID NO:71; nucleotides 25,479 to 25,620 of SEQ ID NO:71; nucleotides 27,398 to 27,555 of SEQ ID NO:71; nucleotides 30,753 to 30,895 of SEQ ID NO:71; nucleotides 30,966 to 31,071 of SEQ ID NO:71; nucleotides 34,199 to 34,315 of SEQ ID NO:71; nucleotides 34,378 to 34,492 of SEQ ID NO:71; nucleotides 36,534 to 36,660 of SEQ ID NO:71; nucleotides 36,737 to 36,939 of SEQ ID NO:71; nucleotides 37,396 to 37,528 of SEQ ID NO:71; nucleotides 40,069 to 40,303 of SEQ ID NO:71; nucleotides 43,559 to 43,811 of SEQ ID NO:71; nucleotides 46,951 to 47,038 of SEQ ID NO:71; nucleotides 47,874 to 48,074 of SEQ ID NO:71; nucleotides 57,044 to 57,276 of SEQ ID NO:71; nucleotides 60,486 to 60,642 of SEQ ID NO:71; nucleotides 62,584 to 62,792 of SEQ ID NO:71; nucleotides 63,526 to 63,681 of SEQ ID NO:71; nucleotides 64,899 to 65,018 of SEQ ID NO:71; nucleotides 69,451 to 69,627 of SEQ ID NO:71; nucleotides 70,604 to 70,936 of SEQ ID NO:71; nucleotides 76,649 to 76,916 of SEQ ID NO:71; nucleotides 77,991 to 78,118 of SEQ ID NO:71; nucleotides 79,337 to 79,626 of SEQ ID NO:71; nucleotides 79,894 to 80,076 of SEQ ID NO:71; nucleotides 81,569 to 81,743 of SEQ ID NO:71; nucleotides 83,354 to 83,756 of SEQ ID NO:71; nucleotides 1 to 401 of SEQ ID NO:72; nucleotides 13,430 to 13,576 of SEQ ID NO:72; nucleotides 14,783 to 14,848 of SEQ ID NO:72; nucleotides 17,228 to 17,323 of SEQ ID NO:72; nucleotides 18,072 to 18,354 of SEQ ID NO:72; nucleotides 34,860 to 34,991 of SEQ ID NO:72; nucleotides 36,100 to 36,277 of SEQ ID NO:72; nucleotides 37,700 to 37,915 of SEQ ID NO:72; nucleotides 40,657 to 40,825 of SEQ ID NO:72; nucleotides 45,298 to 45,516 of SEQ ID NO:72; nucleotides 51,603 to 51,760 of SEQ ID NO:72; nucleotides 54,895 to 55,049 of SEQ ID NO:72; nucleotides 57,742 to 57,911 of SEQ ID NO:72; nucleotides 59,532 to 59,779 of SEQ ID NO:72; nucleotides 61,367 to 61,581 of SEQ ID NO:72; nucleotides 62,031 to 62,276 of SEQ ID NO:72; nucleotides 63,092 to 63,199 of SEQ ID NO:72; nucleotides 67,274 to 67,420 of SEQ ID NO:72; nucleotides 73,454 to 73,662 of SEQ ID NO:72; nucleotides 86,621 to 86,769 of SEQ ID NO:72; nucleotides 86,816 to 87,021 of SEQ ID NO:72; nucleotides 95,149 to 95,396 of SEQ ID NO:72; nucleotides 96,157 to 96,442 of SEQ ID NO:72; nucleotides 99,049 to 99,249 of SEQ ID NO:72; nucleotides 100,720 to 100,961 of SEQ ID NO:72; nucleotides 104,220 to 104,470 of SEQ ID NO:72; nucleotides 106,779 to 106,969 of SEQ ID NO:72; nucleotides 107,760 to 108,687 of SEQ ID NO:72; nucleotides 1 to 1,802 of SEQ ID NO:73; nucleotides 1,803 to 2,186 of SEQ ID NO:73; nucleotides 2,299 to 2,400 of SEQ ID NO:73; nucleotides 3,075 to 3,254 of SEQ ID NO:73; nucleotides 3,611 to 3,760 of SEQ ID NO:73; nucleotides 3,879 to 4,008 of SEQ ID NO:73; nucleotides 6,675 to 6,773 of SEQ ID NO:73; nucleotides 16,442 to 16,636 of SEQ ID NO:73; nucleotides 24,758 to 24,859 of SEQ ID NO:73; nucleotides 26,215 to 26,450 of SEQ ID NO:73; nucleotides 27,556 to 27,771 of SEQ ID NO:73; nucleotides 29,258 to 29,426 of SEQ ID NO:73; nucleotides 32,145 to 32,360 of SEQ ID NO:73; nucleotides 34,375 to 34,532 of SEQ ID NO:73; nucleotides 35,577 to 35,731 of SEQ ID NO:73; nucleotides 35,906 to 36,075 of SEQ ID NO:73; nucleotides 38,948 to 39,195 of SEQ ID NO:73; nucleotides 39,513 to 39,727 of SEQ ID NO:73; nucleotides 47,785 to 47,990 of SEQ ID NO:73; nucleotides 52,430 to 52,644 of SEQ ID NO:73; nucleotides 55,298 to 55,488 of SEQ ID NO:73; nucleotides 57,125 to 57,273 of SEQ ID NO:73; nucleotides 59,275 to 59,396 of SEQ ID NO:73; nucleotides 60,730 to 60,857 of SEQ ID NO:73; nucleotides 61,835 to 62,082 of SEQ ID NO:73; nucleotides 65,633 to 65,777 of SEQ ID NO:73; nucleotides 67,317 to 67,501 of SEQ ID NO:73; nucleotides 69,958 to 70,158 of SEQ ID NO:73; nucleotides 71,116 to 71,357 of SEQ ID NO:73; nucleotides 72,835 to 73,106 of SEQ ID NO:73; nucleotides 76,069 to 76,259 of SEQ ID NO:73; nucleotides 77,048 to 77,349 of SEQ ID NO:73; nucleotides 1 to 1,015 of SEQ ID NO:74; nucleotides 1,415 to 1,764 of SEQ ID NO:74; nucleotides 6,490 to 6,689 of SEQ ID NO:74; nucleotides 8,080 to 8,239 of SEQ ID NO:74; nucleotides 10,407 to 10,502 of SEQ ID NO:74; nucleotides 15,699 to 15,997 of SEQ ID NO:74; nucleotides 17,963 to 18,067 of SEQ ID NO:74; nucleotides 21,823 to 21,997 of SEQ ID NO:74; nucleotides 22,171 to 22,334 of SEQ ID NO:74; nucleotides 22,727 to 22,884 of SEQ ID NO:74; nucleotides 24,061 to 24,218 of SEQ ID NO:74; nucleotides 24,786 to 24,943 of SEQ ID NO:74; nucleotides 25,314 to 25,404 of SEQ ID NO:74; nucleotides 25,699 to 25,916 of SEQ ID NO:74; nucleotides 26,311 to 26,505 of SEQ ID NO:74; nucleotides 27,318 to 27,526 of SEQ ID NO:74; nucleotides 28,337 to 28,494 of SEQ ID NO:74; nucleotides 28,952 to 29,109 of SEQ ID NO:74; nucleotides 29,165 to 29,322 of SEQ ID NO:74; nucleotides 29,442 to 29,602 of SEQ ID NO:74; nucleotides 30,037 to 30,108 of SEQ ID NO:74; nucleotides 30,558 to 30,687 of SEQ ID NO:74; nucleotides 31,820 to 31,977 of SEQ ID NO:74; nucleotides 32,529 to 32,682 of SEQ ID NO:74; nucleotides 32,728 to 33,005 of SEQ ID NO:74; nucleotides 33,177 to 33,352 of SEQ ID NO:74; nucleotides 33,488 to 33,682 of SEQ ID NO:74; nucleotides 34,466 to 36,779 of SEQ ID NO:74; nucleotides 1 to 538 of SEQ ID NO:75; nucleotides 539 to 792 of SEQ ID NO:75; nucleotides 886 to 1,033 of SEQ ID NO:75; nucleotides 6,308 to 7,001 of SEQ ID NO:75; nucleotides 8,408 to 8,722 of SEQ ID NO:75; nucleotides 9,938 to 10,129 of SEQ ID NO:75; or nucleotides 10,909 to 11,232 of SEQ ID NO:75.
Methods of using and analyzing microarrays are known in the art. Hybridization and wash protocols are described extensively in GeneChip® CustomSeqTM Resequencing Array Protocol, (2004) V.2.0, revision 3, Affymetrix. Microarray analysis is discussed extensively in Cutler et al. (2001) Genome Res 11: 1913-1925 and GeneChip® CustomSeqTM Resequencing Array Protocol, (2004) V.2.0, revision 3, Affymetrix, herein incorporated by reference in their entirety.
In an embodiment, the invention provides kits for performing the methods of the invention. Such kits comprise an isolated nucleic acid molecule of the invention, particularly multiple isolated nucleic acid molecules of the invention, and an oligonucleotide pair of the invention, particularly multiple oligonucleotide pairs of the invention. A kit of the invention may further comprise a cholestasis related gene resequencing microarray. Additionally a kit of the invention may comprise a standard sequence profile or a means of accessing a standard sequence profile.
By “standard sequence profile” is intended a listing of the consensus nucleotide sequence of at least one nucleotide sequence of interest. A standard sequence profile may contain additional information including but not limited to, a correlation of at least one nucleotide sequence variation with a disease, syndrome, prognosis, or treatment protocol. A standard sequence profile may exist in printed or electronic form, a remotely accessible form, in a database, or in any other means known in the art. In an embodiment a standard sequence profile is a cholestasis related standard sequence profile. For instance, an exemplary cholestasis related standard sequence profile correlates certain nucleotide alterations in ATP8B1 with either PFIC Type I or BRIC Type 1; ABCB11 with either PFIC Type II or BRIC Type II; ABCB4 with PFIC Type III or MDR deficiency; JAG1 with Alagille Syndrome; or SERPINA-1 with alpha1-antitrypsin deficiency.
By “disease classification”, “disease type”, or “disease subtype” is intended a set of diseases limited by certain shared characteristics, phenotypes, genotypes, or traits. Disease classification for various diseases and disorders are known in the art. Typically cholestasis related syndromes are classified based on the location of cause of the cholestasis, that is, within the liver or outside the liver.
By “subject” is intended a human, human tissue, or human cells. Suitable subjects include, but are not limited to, humans exhibiting cholestasis, tissue obtained from a human exhibiting cholestasis, cells obtained from a human exhibiting cholestasis, cells cultured from a human exhibiting cholestasis, and healthy humans.
By “biological sample” is intended a sample collected from a subject including, but not limited to, whole blood, tissue, cells, mucosa, fluid, scrapings, hairs, cell lysates, and secretions. Biological samples such as blood samples can be obtained by any method known to one skilled in the art. Further, biological samples can be enriched, purified, isolated, or stabilized by any method known to one skilled in the art.
The invention encompasses isolated or substantially purified nucleic acid or protein compositions.
By “disease progression” or “disease course” is intended the physiological events related to a disease or disorder that occur during the period after the initial presentation in a subject or patient exhibiting a disease or disorder. With respect to cholestasis, the duration and degree of the cholestasis are used to describe the disease course or progression. For instance, benign recurrent intrahepatic cholestasis presents and regresses, typically without the development of cirrhosis. Progressive familial intrahepatic cholestasis presents early in infancy and typically continues with the development of cirrhosis and eventual lethality. Identifying the disease course or disease progression that an untreated patient would experience allows practitioners to identify those patients who would benefit most from aggressive early intervention.
The following examples are offered by way of illustration and not limitation.
EXPERIMENTAL
Example 1
Enzymatic Amplification of Multiple Regions of Cholestasis Related Genes
Genomic DNA was purified from whole blood samples obtained from subjects exhibiting jaundice. An enzymatic amplification master mix was prepared following the polymerase manufacturer's standard protocol. The master mix was aliquoted into thermocycler tubes. The isolated nucleic acid molecules of the invention were diluted and added in pairs to each tube. The purified genomic DNA was diluted and added into each tube. The reaction mixtures were placed in a thermocycler and subjected to incubations suitable for long-range PCR. Aliquots of each reaction mixture were loaded on agarose gels and subjected to electrophoresis. Typical results obtained with oligonucleotide pairs specific to ATP8B1 are shown in FIG. 1, Panel C; ABCB11 in FIG. 2, Panel C; ABCB4 in FIG. 3, Panel C; JAG1 in FIG. 4, Panel C; and SERPINA-1 in FIG. 5, Panel C.
Example 2
Resequencing Microarray Analysis
Amplified DNA obtained as described above was utilized in resequencing microarray analysis with a cholestasis related gene resequencing microarray of the invention. Microarray analysis was performed according to the manufacturer's recommended protocol.
All publications, patents, and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications, patents, and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

## Claims

1. An oligonucleotide pair library comprising at least one oligonucleotide pair, said oligonucleotide pair comprising a first nucleic acid molecule and a second nucleic acid molecule wherein said oligonucleotide pair is selected from the group consisting of:
(a) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:1 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:2;
(b) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:3 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:4;
(c) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:5 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:6;
(d) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:7 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:8;
(e) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:9 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:10;
(f) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:11 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:12;
(g) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:13 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:14;
(h) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:15 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:16;
(i) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:17 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:18;
(j) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:19 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:20;
(k) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:21 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:22;
(l) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:23 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:24;
(m) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:25 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:26;
(n) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:27 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:28;
(o) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:29 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:30;
(p) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:31 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:32;
(q) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:33 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:34;
(r) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:35 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:36;
(s) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:37 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:38;
(t) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:39 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:40;
(u) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:41 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:42;
(v) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:43 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:44;
(w) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:45 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:46;
(x) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:47 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:48;
(y) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:49 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:50;
(z) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:51 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:52;
(aa) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:53 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:54;
(ab) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:55 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:56;
(ac) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:57 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:58;
(ad) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:59 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:60;
(ae) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:61 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:62;
(af) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:63 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:64;
(ag) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:65 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:66;
(ah) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:67 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:68;
(ai) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:69 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:70;
(aj) an oligonucleotide pair comprising a first nucleic acid molecule having a nucleotide sequence that differs by one nucleotide alteration from that set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, or SEQ ID NO:69; and
(ak) an oligonucleotide pair comprising a second nucleic acid molecule having a nucleotide sequence that differs by one nucleotide alteration from that set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, or SEQ ID NO:70.

2. The oligonucleotide pair library of claim 1, wherein said first and second nucleic acid molecules of said oligonucleotide pair, allow amplification of a region of the ATP8B1 protein gene (SEQ ID NO:71) and wherein said oligonucleotide pair is selected from the group consisting of:
(a) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:1 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:2;
(b) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:3 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:4;
(c) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:5 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:6;
(d) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:7 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:8;
(e) the oligonucleotide pair consisting of a first, nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:9 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:10;
(f) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:11 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:12;
(g) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:13 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:14;
(h) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:15 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:16; and
(i) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:17 and a second nucleic, acid molecule having the nucleotide sequence set forth in SEQ ID NO:18.

3. The oligonucleotide pair library of claim 1, wherein said, first and second nucleic acid molecules of said oligonucleotide pair allow amplification of a region of the ABCB11 protein gene (SEQ ID NO:72) and wherein said oligonucleotide pair is selected from the group consisting of:
(a) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:19 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:20;
(b) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:21 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:22;
(c) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:23 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:24;
(d) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:25 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:26;
(e) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:27 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:28;
(f) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:29 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:30;
(g) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:31 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:32;
(h) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:33 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:34;
(i) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:35 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:36; and
(j) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:37 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:38.

4. The oligonucleotide pair library of claim 1, wherein said first and second nucleic acid molecules of said oligonucleotide pair allow amplification of a region of the ABCB4 protein gene (SEQ ID NO:73) wherein said oligonucleotide pair is selected from the group consisting of:
(a) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:39 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:40;
(b) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:41 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:42;
(c) the oligonucleotide, pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:43 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:44;
(d) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:45 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:46;
(e) the oligonucleotide pair consisting of a first nucleic acid molecule nucleotide sequence set forth in SEQ ID NO:47 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:48;
(f) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:49 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:50;
(g) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:51 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:52;
(h) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:53 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:54; and
(i) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:55 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:56.

5. The oligonucleotide pair library of claim 1, wherein said first and second nucleic acid molecules of said oligonucleotide pair allow amplification of a region of the JAG1 protein gene (SEQ ID NO:74) and wherein said oligonucleotide pair is selected from the group consisting of:
(a) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:57 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:58;
(b) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:59 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:60;
(c) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set, forth in SEQ ID NO:61 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:62;
(d) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:63 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:64; and
(e) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:65 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:66.

6. The oligonucleotide pair library of claim 1, wherein said first and second nucleic acid molecules of said oligonucleotide pair allow amplification of a region of the SERPINA1 protein gene (SEQ ID NO:75) and wherein said oligonucleotide pair is selected from the group consisting of:
(a) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:67 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:68; and
(b) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:69 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:70.

7. A kit comprising a cholestasis related gene resequencing microarray and at least one oligonucleotide pair selected from the group consisting of:
(a) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:1 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:2;
(b) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:3 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:4;
(c) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:5 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:6;
(d) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:7 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:8;
(e) the oligonucleotide pair consisting of a first nucleic acid molecule haying the nucleotide sequence set forth in SEQ ID NO:9 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:10;
(f) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:11 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:12;
(g) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:13 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:14;
(h) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:15 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:16;
(i) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:17 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:18;
(j) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:19 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:20;
(k) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:21 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:22;
(l) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:23 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:24;
(m) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:25 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:26;
(n) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:27 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:28;
(o) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:29 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:30;
(p) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:31 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:32;
(q) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:33 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:34;
(r) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:35 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:36;
(s) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:37 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:38;
(t) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:39 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:40;
(u) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:41 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:42;
(v) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:43 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:44;
(w) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:45 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:46;
(x) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:47 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:48;
(y) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:49 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:50;
(z) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:51 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:52;
(aa) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:53 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:54;
(ab) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:55 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:56;
(ac) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:57 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:58;
(ad) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:59 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:60;
(ae) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:61 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:62;
(af) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:63 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:64;
(ag) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:65 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:66;
(ah) the oligonucleotide pair consisting Of a first, nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:67 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:68;
(ai) the oligonucleotide pair consisting of a first nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:69 and a second nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:70;
(aj) an oligonucleotide pair comprising a first nucleic acid molecule having a nucleotide sequence that differs by one nucleotide alteration from that set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, or SEQ ID NO:69; and
(ak) an oligonucleotide pair comprising a second nucleic acid molecule having a nucleotide sequence that differs by one nucleotide alteration from that set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, or SEQ ID NO:70.