# Patent

## Title
Cystic fibrosis gene mutations

## Abstract

The present invention provides novel mutations of the CFTR gene related to cystic fibrosis or to conditions associated with cystic fibrosis. Also provided are probes for detecting the mutant sequences. Methods of identifying if an individual has a genotype containing one or more mutations in the CFTR gene are further provided.

## Background
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application is a divisional of U.S. application Ser. No. 12/015,467, filed Jan. 16, 2008 which is a divisional of U.S. application Ser. No. 11/074,903, filed Mar. 7, 2005, which claims the benefit of U.S. Provisional Application No. 60/550,989, filed Mar. 5, 2004, each of which are incorporated herein by reference in its entirety.


FIELD OF THE INVENTION
The present invention relates to novel cystic fibrosis transmembrane regulator (CFTR) gene mutations and for detecting the presence of these mutations in the CFTR gene of individuals.
BACKGROUND OF THE INVENTION
The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.
Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disorder in the Caucasian population. It affects approximately 1 in 2,500 live births in North America (Boat et al, The Metabolic Basis of Inherited Disease, 6th ed, pp 2649-2680, McGraw Hill, NY (1989)). Approximately 1 in 25 persons are carriers of the disease. The responsible gene has been localized to a 250,000 base pair genomic sequence present on the long arm of chromosome 7. This sequence encodes a membrane-associated protein called the “cystic fibrosis transmembrane regulator” (or “CFTR”). There are greater than 1000 different mutations in the CFTR gene, having varying frequencies of occurrence in the population, presently reported to the Cystic Fibrosis Genetic Analysis Consortium. These mutations exist in both the coding regions (e.g., ΔF508, a mutation found on about 70% of CF alleles, represents a deletion of a phenylalanine at residue 508) and the non-coding regions (e.g., the 5T, 7T, and 9T mutations correspond to a sequence of 5, 7, or 9 thymidine bases located at the splice branch/acceptor site of intron 8) of the CFTR gene.
The major symptoms of cystic fibrosis include chronic pulmonary disease, pancreatic exocrine insufficiency, and elevated sweat electrolyte levels. The symptoms are consistent with cystic fibrosis being an exocrine disorder. Although recent advances have been made in the analysis of ion transport across the apical membrane of the epithelium of CF patient cells, it is not clear that the abnormal regulation of chloride channels represents the primary defect in the disease.
A variety of CFTR gene mutations are known. The identity of additional mutations will further assist in the diagnosis of cystic fibrosis.
SUMMARY OF THE INVENTION
The inventors have discovered new mutations in the CFTR gene. These mutations, include 605G->C, 1198-1203del/1204G->A (deletes TGGGCT and replaces G with A at position 1204), 1484G->T, 1573A->G, 1604G->C, 1641-1642AG->T, 2949-2953del (deletes TACTC), 2978A->T, 3239C->A, and 3429C->A, are related to the function of the CFTR gene and, therefore, to cystic fibrosis. These mutations are associated with cystic fibrosis or are associated with conditions associated with cystic fibrosis. By “conditions associated with cystic fibrosis” is meant any clinical symptoms that may be found in a cystic fibrosis patient and are due to one or more CF mutations.
Accordingly, in one aspect, the present invention provides a method of determining if a CFTR gene contains one or more mutations selected from the group consisting of 605G->C, 1198-1203del/1204G->A (deletes TGGGCT and replaces G with A at position 1204), 1484G->T, 1573A->G, 1604G->C, 1641-1642AG->T, 2949-2953del (deletes TACTC), 2978A->T, 3239C->A, and 3429C->A, comprising determining if CFTR nucleic acid contains one or more of the mutations.
In another aspect, the present invention provides a method of identifying if an individual has one or more mutations in the CFTR gene comprising determining if nucleic acid from the individual has one more mutations in one or both CFTR genes, the mutations selected from the group consisting of 605G->C, 1198-1203del/1204G->A (deletes TGGGCT and replaces G with A at position 1204), 1484G->T, 1573A->G, 1604G->C, 1641-1642AG->T, 2949-2953del (deletes TACTC), 2978A->T, 3239C->A, and 3429C->A.
In yet another aspect, the present invention provides a method of determining if an individual is predisposed to cystic fibrosis or to a condition associated with cystic fibrosis comprising determining if nucleic acid from the individual has one more mutations in one or both CFTR genes, the mutations selected from the group consisting of 605G->C, 1198-1203del/12040->A (deletes TGGGCT and replaces G with A at position 1204), 14840->T, 1573A->G, 1604G->C, 1641-1642AG->T, 2949-2953del (deletes TACTC), 2978A->T, 3239C->A, and 3429C->A.
In still a further aspect, the present invention provides a method of counseling an individual on the likelihood of having an offspring afflicted with cystic fibrosis or a condition associated with cystic fibrosis, comprising determining if nucleic acid from the individual has one or more mutations in one or both CFTR genes, the mutations selected from the group consisting of 6050->C, 1198-1203del/1204G->A (deletes TGGGCT and replaces G with A at position 1204), 1484G->T, 1573A->G, 16040->C, 1641-1642AG->T, 2949-2953del (deletes TACTC), 2978A->T, 3239C->A, and 3429C->A.
In all of these aspects, the mutations may be 6050->C and 3239C->A. In some embodiments, 1198-1203del (deletes TGGGCT) and the missense 1204G->A may exist separately from the complex allele, 1198-1203del/1204G->A (deletes TGGGCT and replaces G with A at position 1204). In another embodiment, the mutations are selected from the group consisting of 1198-1203del/1204G->A (deletes TGGGCT and replaces G with A at position 1204), 14840->T, 16040->C, 1641-1642AG->T, 2949-2953del (deletes TACTC), 3239C->A, and 3429C->A. In another embodiment the mutations are selected from the group consisting of 6050->C, 1573A->G, and 2978A->T.
In some embodiments, one more mutations are evaluated for both alleles of the CFTR gene in the individual. By this approach the genotype of the individual can be determined at the position of each mutation.
The presence of the mutation in the CFTR gene may be determined by any of a variety of well known methods used to detect single base changes (transitions and/or small deletions/insertions). Thus, genomic DNA may be isolated from the individual and tested for the CF mutations. In another approach, mRNA can be isolated and tested for the CF mutations. Testing may be performed on mRNA or on a cDNA copy.
Genomic DNA or in cDNA may be subject to amplification by the polymerase chain reaction or related methods using primers directed to specific portions of the CFTR gene which contain a mutation to be detected. The sequence of primers suitable for PCR amplification of portions of the CFTR gene in which contain the CF mutations are also provided.
The presence CF mutations can be determined in a nucleic acid by sequencing appropriate portions of the CFTR gene containing the mutations sought to be detected. In another approach. CF mutations that change susceptibility to digestion by one or more endonuclease restriction enzymes may be used to detect the mutations. In another embodiment, the presence of one or more CF mutations can be determined by allele specific amplification. In yet another embodiment, the presence of one or more CF mutations can be determined by primer extension. In yet a further embodiment, the presence of one or more CF mutations can be determined by oligonucleotide ligation. In another embodiment, the presence of one or more CF mutations can be determined by hybridization with a detectably labeled probe containing the mutant CF sequence.
The methods of the invention also may include detection of other CF mutations which are known in the art and which are described herein.
The present invention also provides oligonucleotide probes that are useful for detecting the CF mutations. Accordingly, provided is a substantially purified nucleic acid comprising 8-20 nucleotides fully complementary to a segment of the CFTR gene that is fully complementary to a portion of the CFTR gene and encompasses a mutant CFTR sequence selected from the group consisting of 605G->C, 1204G->A, 1198-1203del (deletes TGGGCT), 1484G->T, 1573A->G, 1604G->C, 1641-1642AG->T, 2949-2953del (deletes TACTC), 2978A->T, 3239C->A, and 3429C->A, or a complementary nucleic acid sequence thereof. In one embodiment, the purified nucleic acid is no more than 50 nucleotides in length. The invention CF mutant probes may be labeled with a detectable label, which may include any of a radioisotope, a dye, a fluorescent molecule, a hapten or a biotin molecule.
In another aspect the present invention provides kits for one of the methods described herein. In various embodiments, the kits contain one or more of the following components in an amount sufficient to perform a method on at least one sample: one or more primers of the present invention, one or more devices for performing the assay, which may include one or more probes that hybridize to a mutant CF nucleic acid sequence, and optionally contain buffers, enzymes, and reagents for performing a method of detecting a genotype of cystic fibrosis in a nucleic acid sample.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a table showing various CFTR mutations and characterizing information.



DETAILED DESCRIPTION OF THE INVENTION
CF mutations and PCR primer pairs for amplifying segments of the CFTR gene containing the mutation are shown in Table 1.







TABLE 1







CF mutations and associated amplification primers












CF Mutation

Nucleotide Change
Forward and


CF
(HGVS
Nucleotide
(HGVS
Reverse PCR


Mutation
Nomenclature)*
Change
Nomenclature)*
Primers





S158T
p.Ser158Thr
605G−>C
c.473G > C
q4e1F and






q4e1R


V358I
p.Val358Ile
1204G−>A
c.1072G > A
q7e3F and






q7e4R


119del6
p.Trp356_Ala366del
1198-1202del
c.1066_1071del
q7e3F and




(deletes

q7e4R




TGGGCT and




results in W356




and A357)


G451V
p.Gly451Val
1484G−>T
c.1352G > T
q9e9F and






q9e11R


K481E
p.Lys481Glu
1573A−>G
c.1441A > G
s10e3F and






s10e2R


C491S
p.Cys491Ser
1604G−>C
c.1472G > C
s10e3F and






s10e2R


K503N +
p.Lys503fs
1641-1642AG−>T
c.1509_1510delinsT
s10e3F and


frameshift

(deletes 1641A

s10e2R




and 1642G and




replaces with T)


2949del5
p.Thr940fs
2949-2953del
c.2817_2821del
q15e3F and




(deletes TACTC)

q15e4R


H949L
p.His949Leu
2978A−>T
c.2846A > T
q15e3F and






q15e4R


T1036N
p.Thr1036Asn
3239C−>A
c.3107C > A
q17ae1F and






q17ae1R


F1099L
p.Phe1099Leu
3429C−>A
c.3297C > A
q17be1F and






q17be1R





*Nomenclature is based on Human Genome Variation Society guidelines as adopted by Cystic Fibrosis Centre at the Hospital for Sick Children in Toronto, Canada and US Cystic Fibrosis Foundation, Bethesda, MD, USA in April 2010






Further information relating to the CF mutations and the CFTR gene are found in FIG. 1. The primers for amplifying segments of the CFTR gene may hybridize to coding or non-coding CFTR sequences under stringent conditions. Preferred primers are those that flank mutant CF sequences. Primers for CF mutations in Table 1 are shown in Table 2.







TABLE 2







Amplification primer sequences for CF mutations








CF



Mutation
Forward and Reverse PCR Primers





S158T
q4e1F: 



(SEQ ID NO: 33)



TGTAAAACGACGGCCAGTaaagtcttgtgttgaaattctcagg



q4e1R: 



(SEQ ID NO: 34)



CAGGAAACAGCTATGACCCAGCTCACTACCTAATTTATGACAT


 


V358I
q7e3F: 


119de16
(SEQ ID NO: 35)



TGTAAAACGACGGCCAGTcttccattccaagatccc



q7e4R: 



(SEQ ID NO: 36)



CAGGAAACAGCTATGACCGCAAAGTTCATTAGAACTGATC


 


G451V
g9e9F: 



(SEQ ID NO: 37)



TGTAAAACGACGGCCAGTtggatcatgggccatgtgc and



g9e11R: 



(SEQ ID NO: 38)



CAGGAAACAGCTATGACCAAAGAGACATGGACACCAAATTAAG


 


K481E
s10e3F: 


C491S
(SEQ ID NO: 39)


K503N +
TGTAAAACGACGGCCAGTagcagagtacctgaaacagga


frame-
sl0e2R: 


shift
(SEQ ID NO: 40)



CAGGAAACAGCTATGACCCATTCACAGTAGCTTACCCA


 


2949del5
q15c3F: 


H949L
(SEQ ID NO: 41)



TGTAAAACGACGGCCAGTggttaagggtgcatgctcttc



q15e4R: 



(SEQ ID NO: 42)



CAGGAAACAGCTATGACCGGCCCTATTGATGGTGGATC


 


T1036N
q17ae1F: 



(SEQ ID NO: 43)



TGTAAAACGACGGCCAGTacactttgtccactttgc



q17ae1R: 



(SEQ ID NO: 44)



CAGGAAACAGCTATGACCAGATGAGTATCGCACATTC


 


F1099L
q17be1F: 



(SEQ ID NO: 45)



TGTAAAACGACGGCCAGTatctattcaaagaatggcac



q17be1R: 



(SEQ ID NO: 46)



CAGGAAACAGCTATGACCGATAACCTATAGAATGCAGC









By “mutations of the CFTR gene” or “mutant CF sequence” is meant one or more CFTR nucleic acid sequences that are associated or correlated with cystic fibrosis. Additional CF mutations are disclosed in Table 3-17 may be correlated with a carrier state, or with a person afflicted with CF. Thus, the nucleic acid may be tested for CF mutations described in any of Tables 1-17. The nucleic acid sequences containing CF mutations are preferably DNA sequences, and are preferably genomic DNA sequences; however, RNA sequences such as mRNA or hnRNA may also contain nucleic acid mutant sequences that are associated with cystic fibrosis.
By “carrier state” is meant a person who contains one CFTR allele that is a mutant CF nucleic acid sequence, but a second allele that is not a mutant CF nucleic acid sequence. CF is an “autosomal recessive” disease, meaning that a mutation produces little or no phenotypic effect when present in a heterozygous condition with a non-disease related allele, but produces a “disease state” when a person is homozygous, i.e., both CFTR alleles are mutant CF nucleic acid sequences.
By “primer” is meant a sequence of nucleic acid, preferably DNA, that hybridizes to a substantially complementary target sequence and is recognized by DNA polymerase to begin DNA replication.
By “substantially complementary” is meant that two sequences hybridize under stringent hybridization conditions. The skilled artisan will understand that substantially complementary sequences need not hybridize along their entire length. In particular, substantially complementary sequences comprise a contiguous sequence of bases that do not hybridize to a target sequence, positioned 3′ or 5′ to a contiguous sequence of bases that hybridize under stringent hybridization conditions to a target sequence.
By “flanking” is meant that a primer hybridizes to a target nucleic acid adjoining a region of interest sought to be amplified on the target. The skilled artisan will understand that preferred primers are pairs of primers that hybridize 3′ from a region of interest, one on each strand of a target double stranded DNA molecule, such that nucleotides may be add to the 3′ end of the primer by a suitable DNA polymerase. Primers that flank mutant CF sequences do not actually anneal to the mutant sequence but rather anneal to sequence that adjoins the mutant sequence.
By “isolated” a nucleic acid (e.g., an RNA, DNA or a mixed polymer) is one which is substantially separated from other cellular components which naturally accompany such nucleic acid. The term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates, oligonucleotides, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.
By “substantially pure” a nucleic acid, represents more than 50% of the nucleic acid in a sample. The nucleic acid sample may exist in solution or as a dry preparation.
By “complement” is meant the complementary sequence to a nucleic acid according to standard Watson/Crick pairing rules. For example, a sequence (SEQ ID NO: 1) 5′-GCGGTCCCAAAAG-3′ has the complement (SEQ ID NO: 2) 5′-CTTTTGGGACCGC-3′. A complement sequence can also be a sequence of RNA complementary to the DNA sequence or its complement sequence, and can also be a cDNA.
By “coding sequence” is meant a sequence of a nucleic acid or its complement, or a part thereof, that can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof. Coding sequences include exons in a genomic DNA or immature primary RNA transcripts, which are joined together by the cell's biochemical machinery to provide a mature mRNA. The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
By “non-coding sequence” is meant a sequence of a nucleic acid or its complement, or a part thereof, that is not transcribed into amino acid in vivo, or where tRNA does not interact to place or attempt to place an amino acid. Non-coding sequences include both intron sequences in genomic DNA or immature primary RNA transcripts, and gene-associated sequences such as promoters, enhancers, silencers, etc.
Nucleic acid suspected of containing mutant CF sequences are amplified using one or more primers that flank the mutations under conditions such that the primers will amplify CFTR fragments containing the mutations, if present. The oligonucleotide sequences in Table 2 are useful for amplifying segments of the CFTR gene which contain the mutations in Table 1. Nucleic acid from an individual also could be tested for CFTR mutations other than those in Table 1. Such mutations include, for example, any of those listed in Tables 4-18. Primers for these latter CFTR mutations include
The method of identifying the presence or absence of mutant CF sequence by amplification can be used to determine whether a subject has a genotype containing one or more nucleotide sequences correlated with cystic fibrosis. The presence of a wildtype or mutant sequence at each predetermined location can be ascertained by the invention methods.
By “amplification” is meant one or more methods known in the art for copying a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification may be exponential or linear. A target nucleic acid may be either DNA or RNA. The sequences amplified in this manner form an “amplicon.” While the exemplary methods described hereinafter relate to amplification using the polymerase chain reaction (“PCR”), numerous other methods are known in the art for amplification of nucleic acids (e.g., isothermal methods, rolling circle methods, etc.). The skilled artisan will understand that these other methods may be used either in place of, or together with, PCR methods.
The nucleic acid suspected of containing mutant CF sequence may be obtained from a biological sample. By “biological sample” is meant a sample obtained from a biological source. A biological sample can, by way of non-limiting example, consist of or comprise blood, sera, urine, feces, epidermal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample and/or chorionic villi. Convenient biological samples may be obtained by, for example, scraping cells from the surface of the buccal cavity. The term biological sample includes samples which have been processed to release or otherwise make available a nucleic acid for detection as described herein. For example, a biological sample may include a cDNA that has been obtained by reverse transcription of RNA from cells in a biological sample.
By “subject” is meant a human or any other animal which contains as CFTR gene that can be amplified using the primers and methods described herein. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. A human includes pre and post natal forms. Particularly preferred subjects are humans being tested for the existence of a CF carrier state or disease state.
By “identifying” with respect to an amplified sample is meant that the presence or absence of a particular nucleic acid amplification product is detected. Numerous methods for detecting the results of a nucleic acid amplification method are known to those of skill in the art.
The present invention provides specific primers that aid in the detection of mutant CF genotype. Such primers enable the amplification of segments of the CFTR gene that are known to contain mutant CF sequence from a nucleic acid containing biological sample. By amplifying specific regions of the CFTR gene, the invention primers facilitate the identification of wildtype or mutant CF sequence at a particular location of the CFTR gene. Primers for amplifying various regions of the CFTR gene include the following: SEQ ID NO: 3, 5′-GCGGTCCCAAAAGGGTCAGTTGTAGGAAGTCACCAAAG-3′ (g4e1F), and SEQ ID NO: 4,5′-GCGGTCCCAAAAGGGTCAGTCGATACAGAATATATGTGCC-3′ (g4e2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 5,5′-GCGGTCCCAAAAGGGTCAGTGAATCATTCAGTGGGTATAAGCAG-3′ (g19i2F), and SEQ ID NO: 6, 5′-GCGGTCCCAAAAGGGTCAGTCTTCAATGCACCTCCTCCC-3′ (q19i3R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 7, 5′-GCGGTCCCAAAAGGGTCAGTAGATACTTCAATAGCTCAGCC-3′ (g7e1F), and SEQ ID NO: 8,5′-GCGGTCCCAAAAGGGTCAGTGGTACATTACCTGTATTTTGTTT-3′ (g7e2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 9, 5′′-GCGGTCCCAAAAGGGTCAGTGTGAATCGATGTGGTGACCA-3′ (s12e1F), and SEQ ID NO: 10, 5′-GCGGTCCCAAAAGGGTCAGTCTGGTTTAGCATGAGGCGGT-3′(s12e1R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 11, 5′-GCGGTCCCAAAAGGGTCAGTTTGGTTGTGCTGTGGCTCCT-3′ (g14be1F), and SEQ ID NO: 12, 5′-GCGGTCCCAAAAGGGTCAGTACAATACATACAAACATAGTGG-3′ (g14be2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 13, 5′-GCGGTCCCAAAAGGGTCAGTGAAAGTATTTATTTTTTCTGGAAC-3′ (q21e1F), and SEQ ID NO: 14 5′-GCGGTCCCAAAAGGGTCAGTGTGTGTAGAATGATGTCAGCTAT-3′ (q21e2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 15, 5′-GCGGTCCCAAAAGGGTCAGTCAGATTGAGCATACTAAAAGTG-3′ (g11 e1F), and SEQ ID NO: 16, 5′-GCGGTCCCAAAAGGGTCAGTTACATGAATGACATTTACAGCA-3′ (g11e2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 17, 5′-GCGGTCCCAAAAGGGTCAGTAAGAACTGGATCAGGGAAGA-3′ (g20e1F), and SEQ ID NO: 18, 5′-GCGGTCCCAAAAGGGTCAGTTCCTTTTGCTCACCTGTGGT-3′ (g20e2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 19, 5′-GCGGTCCCAAAAGGGTCAGTGGTCCCACTTTTTATTCTTTTGC-3′ (q3e2F), and SEQ ID NO: 20 5′-GCGGTCCCAAAAGGGTCAGTTGGTTTCTTAGTGTTTGGAGTTG-3′ (q3e2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 21, 5′-GCGGTCCCAAAAGGGTCAGTTGGATCATGGGCCATGTGC-3′ (g9e9F), and SEQ ID NO: 22, 5′-GCGGTCCCAAAAGGGTCAGTACTACCTTGCCTGCTCCAGTGG-3′ (g9e9R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 23, 5′-GCGGTCCCAAAAGGGTCAGTAGGTAGCAGCTATTTTTATGG-3′ (g13e2F), and SEQ ID NO: 24, 5′-GCGGTCCCAAAAGGGTCAGTTAAGGGAGTCTTTTGCACAA-3′ (g13c2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 25
5′-GCGGTCCCAAAAGGGTCAGTGCAATTTTGGATGACCTTC-3′ (q16i1F), and SEQ ID NO: 26 5′-GCGGTCCCAAAAGGGTCAGTTAGACAGGACTTCAACCCTC-3′ (q16i2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 27, 5′-GCGGTCCCAAAAGGGTCAGTGGTGATTATGGGAGAACTGG-3′ (q10e10F), and SEQ ID NO: 28, 5′-GCGGTCCCAAAAGGGTCAGTATGCTTTGATGACGCTTC-3′ (q10e11R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 29, 5′-GCGGTCCCAAAAGGGTCAGTTTCATTGAAAAGCCCGAC-3′ (q19e12F), and SEQ ID NO: 30, 5′-GCGGTCCCAAAAGGGTCAGTCACCTTCTGTGTATTTTGCTG-3′ (q19e13R) are preferably used together as forward (F) and reverse (R) primers; and SEQ ID NO: 31, 5′-GCGGTCCCAAAAGGGTCAGTAAGTATTGGACAACTTGTTAGTCTC-3′ (q5e12F), and SEQ ID NO: 32, 5′-GCGGTCCCAAAAGGGTCAGTCGCCTTTCCAGTTGTATAATTT-3′ (q5e13R), are preferably used together as forward (F) and reverse (R) primers. These pairs of primers, which may been used in multiplex amplifications, can amplify the regions of the CFTR gene shown in Table 3.







TABLE 3







CFTR Primer Pairs and Amplicon Characteristics










Forward Primer
Reverse Primer
Exon/Intron
Size





g14be1F (SEQ ID NO. 11)
g14be24 (SEQ ID NO. 12)
14b/i14b
149


q5e12F (SEQ ID NO. 31)
q5e13R (SEQ ID NO. 32)
5/i5
165


g20e1F (SEQ ID NO. 17)
g20e2R (SEQ ID NO. 18)
20
194


q16i1F (SEQ ID NO. 25)
q16i2R (SEQ ID NO. 26)
16/i16
200


q10e10F (SEQ ID NO. 27)
q10e11R (SEQ ID NO. 28)
10
204


q21e1F (SEQ ID NO. 13)
q21e2R (SEQ ID NO. 14)
21
215


g11e1F (SEQ ID NO. 15)
g11e2R (SEQ ID NO. 16)
i10/11/i11
240


g7e1F (SEQ ID NO. 7)
g7e2R (SEQ ID NO. 8)
 7
259


g4e1F (SEQ ID NO. 3)
g4e2R (SEQ ID NO. 4)
4/i4
306


q3e2F (SEQ ID NO. 19)
q3e2R (SEQ ID NO. 20)
3/i3
308


q19e12F (SEQ ID NO. 29)
q1913e2R (SEQ ID NO. 30)
i18/19
310


q13e2F (SEQ ID NO. 23)
g13e2R (SEQ ID NO. 24)
13
334


g9e9F (SEQ ID NO. 21)
g9e9R (SEQ ID NO. 22)
i8/9
351


g19i2F (SEQ ID NO. 5)
g19i3R (SEQ ID NO. 6)
i19
389


s12e1F (SEQ ID NO. 9)
s12e1R (SEQ ID NO. 10)
i11/12/i12
465









The nucleic acid to be amplified may be from a biological sample such as an organism, cell culture, tissue sample, and the like. The biological sample can be from a subject which includes any eukaryotic organism or animal, preferably fungi, invertebrates, insects, arachnids, fish, amphibians, reptiles, birds, marsupials and mammals. A preferred subject is a human, which may be a patient presenting to a medical provider for diagnosis or treatment of a disease. The biological sample may be obtained from a stage of life such as a fetus, young adult, adult, and the like. Particularly preferred subjects are humans being tested for the existence of a CF carrier state or disease state.
The sample to be analyzed may consist of or comprise blood, sera, urine, feces, epidermal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample and/or chorionic villi, and the like. A biological sample may be processed to release or otherwise make available a nucleic acid for detection as described herein. Such processing may include steps of nucleic acid manipulation, e.g., preparing a cDNA by reverse transcription of RNA from the biological sample. Thus, the nucleic acid to be amplified by the methods of the invention may be DNA or RNA.
Nucleic acid may be amplified by one or more methods known in the art for copying a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification may be exponential or linear. The sequences amplified in this manner form an “amplicon.” In a preferred embodiment, the amplification by the is by the polymerase chain reaction (“PCR”) (e.g., Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al., European Patent Appln. 50,424; European Patent Appln. 84,796, European Patent Application 258,017, European Patent Appln. 237,362; Mullis, K., European Patent Appln. 201,184; Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al., U.S. Pat. No. 4,683,194). Other known nucleic acid amplification procedures that can be used include, for example, transcription-based amplification systems or isothermal amplification methods (Malek, L. T. et al., U.S. Pat. No. 5,130,238; Davey, C. et al., European Patent Application 329,822; Schuster et al., U.S. Pat. No. 5,169,766; Miller, H. I. et al., PCT appln. WO 89/06700; Kwoh, D. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:1173 (1989); Gingeras, T. R. et al., PCT Application WO 88/10315; Walker, G. T. et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992)). Amplification may be performed to with relatively similar levels of each primer of a primer pair to generate an double stranded amplicon. However, asymmetric PCR may be used to amplify predominantly or exclusively a single stranded product as is well known in the art (e.g. Poddar et al. Molec. And Cell. Probes 14:25-32 (2000)). This can be achieved for each pair of primers by reducing the concentration of one primer significantly relative to the other primer of the pair (e.g. 100 fold difference). Amplification by asymmetric PCR is generally linear. One of ordinary skill in the art would know that there are many other useful methods that can be employed to amplify nucleic acid with the invention primers (e.g., isothermal methods, rolling circle methods, etc.), and that such methods may be used either in place of, or together with, PCR methods. Persons of ordinary skill in the art also will readily acknowledge that enzymes and reagents necessary for amplifying nucleic acid sequences through the polymerase chain reaction, and techniques and procedures for performing PCR, are well known. The examples below illustrate a standard protocol for performing PCR and the amplification of nucleic acid sequences that correlate with or are indicative of cystic fibrosis.
In another aspect, the present invention provides methods of detecting a cystic fibrosis genotype in a biological sample. The methods comprise amplifying nucleic acids in a biological sample of the subject and identifying the presence or absence of one or more mutant cystic fibrosis nucleic acid sequences in the amplified nucleic acid. Accordingly, the present invention provides a method of determining the presence or absence of one or more mutant cystic fibrosis nucleic acid sequences in a nucleic acid containing sample, comprising: contacting the sample with reagents suitable for nucleic acid amplification including one or more pairs of nucleic acid primers flanking one or more predetermined nucleic acid sequences that are correlated with cystic fibrosis, amplifying the predetermined nucleic acid sequence(s), if present, to provide an amplified sample; and identifying the presence or absence of mutant or wildtype sequences in the amplified sample.
One may analyze the amplified product for the presence of absence of any of a number of mutant CF sequences that may be present in the sample nucleic acid. As already discussed, numerous mutations in the CFTR gene have been associated with CF carrier and disease states. For example, a three base pair deletion leading to the omission of a phenylalanine residue in the gene product has been determined to correspond to the mutations of the CF gene in approximately 70% of the patients affected by CF. The table below identifies preferred CF sequences and identifies which of the primer pairs of the invention may be used to amplify the sequence.







TABLE 4







CFTR mutations that may be detected in amplified product


using as the primer pair SEQ ID NO: 19 and 20.










Name
Nucleotide change
Exon
Consequence





297-3C−>T
C to T at 297-3
intron 2
mRNA splicing defect


E56K
G to A at 298
3
Glu to Lys at 56


300delA
Deletion of A at
3
Frameshift



300


W57R
T to C at 301
3
Trp to Arg at 57


W57G
T to G at 301
3
Trp to Gly at 57


W57X(TAG)
G to A at 302
3
Trp to Stop at 57


W57X(TGA)
G to A at 303
3
Trp to Stop at 57


D58N
G to A at 304
3
Asp to Asn at 58


D58G
A to G at 305
3
Asp to Gly at 58


306insA
Insertion of A at
3
Frameshift



306


306delTAGA
deletion of TAGA
3
Frameshift



from 306


E60L
G to A at 310
3
Glu to Leu at 60


E60X
G to T at 310
3
Glu to Stop at 60


E60K
G to A at 310
3
Glu to Lys at 60


N66S
A to G at 328
3
Asn to Ser at 66


P67L
C to T at 332
3
Pro to Leu at 67


K68E
A to G at 334
3
Lys to Glu at 68


K68N
A to T at 336
3
Lys to Asn at 68


A72T
G to A at 346
3
Ala to Thr at 72


A72D
C to A at 347
3
Ala to Asp at 72


347delC
deletion of C at
3
Frameshift



347


R74W
C to T at 352
3
Arg to Trp at 74


R74Q
G to A at 353
3
Arg to Gln at 74


R75X
C to T at 355
3
Arg to Stop at 75


R75L
G to T at 356
3
Arg to Leu at 75


359insT
Insertion of T
3
Frameshift



after 359


360delT
deletion of T at
3
Frameshift



360


W79R
T to C at 367
3
Trp to Arg at 79


W79X
G to A at 368
3
Trp to Stop at 79


G85E
G to A at 386
3
Gly to Glu at 85


G85V
G to T at 386
3
Gly to Val at 85


F87L
T to C at 391
3
Phe to Leu at 87


394delTT
deletion of TT
3
frameshift



from 394


L88S
T to C at 395
3
Leu to Ser at 88


L88X(T−>A)
T to A at 395
3
Leu to Stop at 88


L88X(T−>G)
T to G at 395
3
Leu to Stop at 88


Y89C
A to G at 398
3
Tyr to Cys at 89


L90S
T to C at 401
3
Leu to Ser at 90


G91R
G to A at 403
3
Gly to Arg at 91


405 + 1G−>A
G to A at 405 + 1
intron 3
mRNA splicing defect


405 + 3A−>C
A to C at 405 + 3
intron 3
mRNA splicing defect


405 + 4A−>G
A to G at 405 + 4
intron 3
mRNA splicing defect
















TABLE 5







CFTR mutations that may be detected in amplified product using


as the primer pair SEQ ID NO: 3 and 4.










Name
Nucleotide change
Exon
Consequence





A96E
C to A at 419
4
Ala to Glu at 96


Q98X
C to T at 424
4
Gln to Stop at 98


Q98P
A to C at 425
4
Gln to Pro at 98


Q98R
A to G at 425
4
Gln to Arg at 98


P99L
C to T at 428
4
Pro to Leu at 99


L101X
T to G at 434
4
Leu to Stop at 101


435insA
Insertion of A after 435
4
Frameshift


G103X
G to T at 439
4
Gly to Stop at 103


441delA
deletion of A at 441 and T to
4
Frameshift



A at 486


444delA
deletion of A at 444
4
Frameshift


I105N
T to A at 446
4
Ile to Asn at 105


451del8
deletion of GCTTCCTA from
4
Frameshift



451


S108F
C to T at 455
4
Ser to Phe at 108


457TAT->G
TAT to G at 457
4
Frameshift


Y109N
T to A at 457
4
Tyr to Asn at 109


458delAT
deletion of AT at 458
4
Frameshift


Y109C
A to G at 458
4
Tyr to Cys at 109


460delG
deletion of G at 460
4
Frameshift


D110Y
G to T at 460
4
Asp to Tyr at 110


D110H
G to C at 460
4
Asp to His at 110


D110E
C to A at 462
4
Asp to Glu at 110


P111A
C to G at 463
4
Pro to Ala at 111


P111L
C to T at 464
4
Pro to Leu at 111


ΔE115
3 bp deletion of 475-477
4
deletion of Glu at 115


E116Q
G to C at 478
4
Glu to Gln at 116


E116K
G to A at 478
4
Glu to Lys at 116


R117C
C to T at 481
4
Arg to Cys at 117


R117P
G to C at 482
4
Arg to Pro at 117


R117L
G to T at 482
4
Arg to Leu at 117


R117H
G to A at 482
4
Arg to His at 117


I119V
A to G at 487
4
Iso to Val at 119


A120T
G to A at 490
4
Ala to Thr at 120


Y122X
T to A at 498
4
Tyr to Stop at 122


I125T
T to C at 506
4
Ile to Thr at 125


G126D
G to A at 509
4
Gly to Asp at 126


L127X
T to G at 512
4
Leu to Stop at 127


525delT
deletion of T at 525
4
Frameshift


541del4
deletion of CTCC from 541
4
Frameshift


541delC
deletion of C at 541
4
Frameshift


L137R
T to G at 542
4
Leu to Arg at 137


L137H
T to A at 542
4
Leu to His at 137


L138ins
insertion of CTA, TAC or
4
insertion of leucine at 138



ACT at nucleotide 544, 545



or 546


546insCTA
Insertion of CTA at 546
4
Frameshift


547insTA
insertion of TA after 547
4
Frameshift


H139L
A to T at 548
4
His to Leu at 548


H139R
A to G at 548
4
His to Arg at 139


P140S
C to T at 550
4
Pro to Ser at 140


P140L
C to T at 551
4
Pro to Leu at 140


552insA
Insertion of A after 552
4
Frameshift


A141D
C to A at 554
4
Ala to Asp at 141


556delA
deletion of A at 556
4
Frameshift


557delT
deletion of T at 557
4
Frameshift


565delC
deletion of C at 565
4
Frameshift


H146R
A to G at 569
4
His to Arg at 146 (CBAVD)


574delA
deletion of A at 574
4
Frameshift


I148N
T to A at 575
4
Ile to Asn at 148


I148T
T to C at 575
4
Ile to Thr at 148


G149R
G to A at 577
4
Gly to Arg at 149


Q151X
C to T at 583
4
Gln to Stop at 151


M152V
A to G at 586
4
Met to Val at 152 (mutation)


M152R
T to G at 587
4
Met to Arg at 152


591del18
deletion of 18 bp from 591
4
deletion of 6 amino acids





from the CFTR protein


A155P
G to C at 595
4
Ala to Pro at 155


S158R
A to C at 604
4
Ser to Arg at 158


605insT
Insertion of T after 605
4
Frameshift


L159X
T to A at 608
4
Leu to Stop at 159


Y161D
T to G at 613
4
Tyr to Asp at 161


Y161N
T to A at 613
4
Tyr to Asn at 161


Y161S
A to C at 614 (together with
4
Tyr to Ser at 161



612T/A)


K162E
A to G at 616
4
Lys to Glu at 162


621G->A
G to A at 621
4
mRNA splicing defect


621+1G->T
G to T at 621 + 1
intron 4
mRNA splicing defect


621+2T->C
T to C at 621 + 2
intron 4
mRNA splicing defect


621+2T->G
T to G at 621 + 2
intron 4
mRNA splicing defect


621+3A->G
A to G at 621 + 3
intron 4
mRNA splicing defect
















TABLE 6







CFTR mutations that may be detected in amplified product


using as the primer pair SEQ ID NO: 31 and 32.










Name
Nucleotide_change
Exon
Consequence





681delC
deletion of C at 681
5
Frameshift


N186K
C to A at 690
5
Asn to Lys at 186


N187K
C to A at 693
5
Asn to Lys at 187


ΔD192
deletion of TGA or
5
deletion of Asp at 192



GAT from 706 or 707


D192N
G to A at 706
5
Asp to Asn at 192


D192G
A to G at 707
5
Asp to Gly at 192


E193K
G to A at 709
5
Glu to Lys at 193


E193X
G to T at 709
5
Glu to Stop at 193


711+1G->T
G to T at 711 + 1
intron 5
mRNA splicing defect


711+3A->G
A to G at 711 + 3
intron 5
mRNA splicing defect


711+3A->C
A to C at 711 + 3
intron 5
mRNA splicing defect


711+3A->T
A to T at 711 + 3
intron 5
mRNA splicing defect


711+5G->A
G to A at 711 + 5
intron 5
mRNA splicing defect


711+34A->G
A to G at 711 + 34
intron 5
mRNA splicing defect
















TABLE 7







CFTR mutations that may be detected in amplified product using


as the primer pair SEQ ID NO: 7 and 8.










Name
Nucleotide_change
Exon
Consequence





ΔF311
deletion of 3 bp between 1059
7
deletion of Phe310, 311 or 312



and 1069


F311L
C to G at 1065
7
Phe to Leu at 311


G314R
G to C at 1072
7
Gly to Arg at 314


G314E
G to A at 1073
7
Gly to Glu at 314


G314V
G to T at 1073
7
Gly to Val at 324


F316L
T to G at 1077
7
Phe to Leu at 316


1078delT
deletion of T at 1078
7
Frameshift


V317A
T to C at 1082
7
Val to Ala at 317


L320V
T to G at 1090
7
Leu to Val at 320 CAVD


L320X
T to A at 1091
7
Leu to Stop at 320


L320F
A to T at 1092
7
Leu to Phe at 320


V322A
T to C at 1097
7
Val to Ala at 322


1112delT
deletion of T at 1112
7
Frameshift


L327R
T to G at 1112
7
Leu to Arg at 327


1119delA
deletion of A at 1119
7
Frameshift


G330X
G to T at 1120
7
Gly to Stop at 330


R334W
C to T at 1132
7
Arg to Trp at 334


R334Q
G to A at 1133
7
Arg to Gln at 334


R334L
G to T at 1133
7
Arg to Leu at 334


1138insG
Insertion of G after 1138
7
Frameshift


I336K
T to A at 1139
7
Ile to Lys at 336


T338I
C to T at 1145
7
Thr to Ile at 338


1150delA
deletion of A at 1150
7
Frameshift


1154insTC
insertion of TC after 1154
7
Frameshift


1161insG
Insertion of G after 1161
7
Frameshift


1161delC
deletion of C at 1161
7
Frameshift


L346P
T to C at 1169
7
Leu to Pro at 346


R347C
C to T at 1171
7
Arg to Cys at 347


R347H
G to A at 1172
7
Arg to His at 347


R347L
G to T at 1172
7
Arg to Leu at 347


R347P
G to C at 1172
7
Arg to Pro at 347


M348K
T to A at 1175
7
Met to Lys at 348


A349V
C to T at 1178
7
Ala to Val at 349


R352W
C to T at 1186
7
Arg to Trp at 352


R352Q
G to A at 1187
7
Arg to Gln at 352


Q353X
C to T at 1189
7
Gln to Stp at 353


Q353H
A to C at 1191
7
Gln to His at 353


1199delG
deletion of G at 1199
7
Frameshift


W356X
G to A at 1200
7
Trp to Stop at 356


Q359K/T360K
C to A at 1207 and C to A at
7
Glu to Lys at 359 and Thr to Lys



1211

at 360


Q359R
A to G at 1208
7
Gln to Arg at 359


1213delT
deletion of T at 1213
7
Frameshift


W361R(T->C)
T to C at 1213
7
Trp to Arg at 361


W361R(T->A)
T to A at 1213
7
Trp to Arg at 361


1215delG
deletion of G at 1215
7
Frameshift


1221delCT
deletion of CT from 1221
7
Frameshift


S364P
T to C at 1222
7
Ser to Pro at 364


L365P
T to C at 1226
7
Leu to Pro at 365
















TABLE 8







CFTR mutations that may be detected in amplified product using


as the primer pair SEQ ID NO: 21 and 22.










Name
Nucleotide_change
Exon
Consequence





1342-11TTT->G
TTT to G at 1342-11
intron 8
mRNA splicing defect


1342-2delAG
deletion of AG from 1342-2
intron 8
Frameshift


1342-2A->C
A to C at 1342-2
intron 8
mRNA splicing defect


1342-1G->C
G to C at 1342-1
intron 8
mRNA splicing defect


E407V
A to T at 1352
9
Glu to Val at 407


1366delG
deletion of G at 1366
9
Frameshift


1367delC
deletion of C at 1367
9
Frameshift


1367del5
deletion of CAAAA at 1367
9
Frameshift


Q414X
C to T at 1372
9
Gln to Stop at 414


N418S
A to G at 1385
9
Asn to Ser at 418


G424S
G to A at 1402
9
Gly to Ser at 424


S434X
C to G at 1433
9
Ser to Stop at 434


D443Y
G to T at 1459
9
Asp to Tyr at 443


1460delAT
deletion of AT from 1460
9
Frameshift


1461ins4
insertion of AGAT after 1461
9
Frameshift


I444S
T to G at 1463
9
Ile to Ser at 444


1471delA
deletion of A at 1471
9
Frameshift


Q452P
A to C at 1487
9
Gln to Pro at 452


ΔL453
deletion of 3 bp between 1488
9
deletion of Leu at 452 or 454



and 1494


A455E
C to A at 1496
9
Ala to Glu at 455


V456F
G to T at 1498
9
Val to Phe at 456
















TABLE 9







CFTR mutations that may be detected in amplified product using


as the primer pair SEQ ID NO: 27 and 28.










Name
Nucleotide_change
Exon
Consequence





G480C
G to T at 1570
10
Gly to Cys at 480


G480D
G to A at 1570
10
Gly to Asp at 480


G480S
G to A at 1570
10
Gly to Ser at 480


1571delG
deletion of G at 1571
10
Frameshift


1576insT
Insertion of T at 1576
10
Frameshift


H484Y
C to T at 1582
10
His to Tyr at 484 (CBAVD)


H484R
A to G at 1583
10
His to Arg at 484


S485C
A to T at 1585
10
Ser to Cys at 485


G486X
G to T at 1588
10
Glu to Stop at 486


S489X
C to A at 1598
10
Ser to Stop at 489


1601delTC
deletion of TC from 1601 or
10
Frameshift



CT from 1602


C491R
T to C at 1603
10
Cys to Arg at 491


S492F
C to T at 1607
10
Ser to Phe at 492


Q493X
C to T at 1609
10
Gln to Stop at 493


1609delCA
deletion of CA from 1609
10
Frameshift


Q493R
A to G at 1610
10
Gln to Arg at 493


1612delTT
deletion of TT from 1612
10
Frameshift


W496X
G to A at 1619
10
Trp to Stop at 496


P499A
C to G at 1627
10
Pro to Ala at 499 (CBAVD)


T501A
A to G at 1633
10
Thr to Ala at 501


I502T
T to C at 1637
10
Ile to Thr at 502


I502N
T to A at 1637
10
Ile to Asn at 502


E504X
G to T at 1642
10
Glu to Stop at 504


E504Q
G to C at 1642
10
Glu to Gln at 504


I506L
A to C at 1648
10
Ile to Leu at 506


ΔI507
deletion of 3 bp between 1648
10
deletion of Ile506 or Ile507



and 1653


I506S
T to G at 1649
10
Ile to Ser at 506


I506T
T to C at 1649
10
Ile to Thr at 506


ΔF508
deletion of 3 bp between 1652
10
deletion of Phe at 508



and 1655


F508S
T to C at 1655
10
Phe to Ser at 508


D513G
A to G at 1670
10
Asp to Gly at 513 (CBAVD)


1677delTA
deletion of TA from 1677
10
frameshift


Y517C
A to G at 1682
10
Tyr to Cys at 517
















TABLE 10







CFTR mutations that may be detected in amplified product using


as the primer pair SEQ ID NO: 15 and 16.










Name
Nucleotide_change
Exon
Consequence





1716-1G->A
G to A at 1716-1
intron 10
mRNA splicing defect


1717-8G->A
G to A at 1717-8
intron 10
mRNA splicing defect


1717-3T->G
T to G at 1717-3
intron 10
mRNA splicing defect


1717-2A->G
A to G at 1717-2
intron 10
mRNA splicing defect


1717-1G->A
G to A at 1717-1
intron 10
mRNA splicing defect


D529H
G to C at 1717
11
Asp to His at 529


1717-9T->A
T to A at 1717-9
intron 10
mRNA splicing mutation


A534E
C to A at 1733
11
Ala to Glu at 534


1742delAC
deletion of AC from 1742
11
Frameshift


I539T
T to C at 1748
11
Ile to Thr at 539


1749insTA
Insertion of TA at 1749
11
frameshift resulting in





premature termination at 540


G542X
G to T at 1756
11
Gly to Stop at 542


G544S
G to A at 1762
11
Gly to Ser at 544


G544V
G to T at 1763
11
Gly to Val at 544 (CBAVD)


1774delCT
deletion of CT from 1774
11
Frameshift


S549R(A->C)
A to C at 1777
11
Ser to Arg at 549


S549I
G to T at 1778
11
Ser to Ile at 549


S549N
G to A at 1778
11
Ser to Asn at 549


S549R(T->G)
T to G at 1779
11
Ser to Arg at 549


G550X
G to T at 1780
11
Gly to Stop at 550


G550R
G to A at 1780
11
Gly to Arg at 550


1782delA
deletion of A at 1782
11
Frameshift


G551S
G to A at 1783
11
Gly to Ser at 551


1784delG
deletion of G at 1784
11
Frameshift


G551D
G to A at 1784
11
Gly to Asp at 551


Q552X
C to T at 1786
11
Gln to Stop at 552


Q552K
C to A at 1786
11
Gln to Lys


1787delA
deletion of A at position
11
frameshift, stop codon at 558



1787 or 1788


R553G
C to G at 1789
11
Arg to Gly at 553


R553X
C to T at 1789
11
Arg to Stop at 553


R553Q
G to A at 1790
11
Arg to Gln at 553 (associated





with ΔF508);


R555G
A to G at 1795
11
Arg to Gly at 555


I556V
A to G at 1798
11
Ile to Val at 556


1802delC
deletion of C at 1802
11
Frameshift


L558S
T to C at 1805
11
Leu to Ser at 558


1806delA
deletion of A at 1806
11
Frameshift


A559T
G to A at 1807
11
Ala to Thr at 559


A559E
C to A at 1808
11
Ala to Glu at 559


R560T
G to C at 1811
11
Arg to Thr at 560; mRNA





splicing defect


R560K
G to A at 1811
11
Arg to Lys at 560


1811+1G->C
G to C at 1811 + 1
intron 11
mRNA splicing defect


1811+1.6kbA->G
A to G at 1811 + 1.2kb
intron 11
creation of splice donor site


1811+18G->A
G to A at 1811 + 18
intron 11
mRNA splicing defect
















TABLE 11







CFTR mutations that may be detected in amplified product using as the primer pair


SEQ ID NO: 9 and 10.










Name
Nucleotide change
Exon
Consequence





1812-1G->A
G to A at 1812-1
intron 11
mRNA splicing defect


R560S
A to C at 1812
12
Arg to Ser at 560


1813insC
Insertion of C after 1813 (or
12
Frameshift



1814)


A561E
C to A at 1814
12
Ala to Glu at 561


V562I
G to A at 1816
12
Val to Ile at 562


V562L
G to C at 1816
12
Val to Leu at 562


Y563D
T to G at 1819
12
Tyr to Asp at 563


Y563N
T to A at 1819
12
Tyr to Asn at 563


Y563C
A to G at 1821
12
Tyr to Cys at 563


1833delT
deletion of T at 1833
12
Frameshift


L568X
T to A at 1835
12
Leu to Stop at 568


L568F
G to T at 1836
12
Leu to Phe at 568 (CBAVD)


Y569D
T to G at 1837
12
Tyr to Asp at 569


Y569H
T to C at 1837
12
Tyr to His at 569


Y569C
A to G at 1838
12
Tyr to Cys at 569


V569X
T to A at 1839
12
Tyr to Stop at 569


L571S
T to C at 1844
12
Leu to Ser at 571


1845delAG/1846delGA
deletion of AG at 1845 or
12
Frameshift



GA at 1846


D572N
G to A at 1846
12
Asp to Asn at 572


P574H
C to A at 1853
12
Pro to His at 574


G576X
G to T at 1858
12
Gly to Stop at 576


G576A
G to C at 1859
12
Gly to Ala at 576 (CAVD)


Y577F
A to T at 1862
12
Tyr to Phe at 577


D579Y
G to T at 1867
12
Asp to Tyr at 579


D579G
A to G at 1868
12
Asp to Gly at 579


D579A
A to C at 1868
12
Asp to Ala at 579


1870delG
deletion of T between 1871
12
Frameshift


1874insT
Insertion of T between 1871
12
Frameshift



and 1874


T582R
C to G at 1877
12
Thr to Arg at 582


T582I
C to T at 1877
12
Thr to Ile at 582


E585X
G to T at 1885
12
Glu to Stop at 585


S589N
G to A at 1898
12
Ser to Asn at 589 (mRNA





splicing defect)


S589I
G to T at 1898
12
Ser to Ile at 589 (splicing)


1898+1G->A
G to A at 1898 + 1
intron 12
mRNA splicing defect


1898+1G->C
G to C at 1898 + 1
intron 12
mRNA splicing defect


1898+1G->T
G to T at 1898 + 1
intron 12
mRNA splicing defect


1898+3A->G
A to G at 1898 + 3
intron 12
mRNA splicing defect


1898+3A->C
A to C at 1898 + 3
intron 12
mRNA splicing defect


1898+5G->A
G to A at 1898 + 5
intron 12
mRNA splicing defect


1898+5G->T
G to T at 1898 + 5
intron 12
mRNA splicing defect


1898+73T->G
T to G at 1898 + 73
intron 12
mRNA splicing defect
















TABLE 12







CFTR mutations that may be detected in amplified product using


as the primer pair SEQ ID NO: 23 and 24.










Name
Nucleotide_change
Exon
Consequence





1918delGC
deletion of GC from 1918
13
Frameshift


1924del7
deletion of 7 bp (AAACTA)
13
Frameshift



from 1924


R600G
A to G at 1930
13
Arg to Gly at 600


I601F
A to T at 1933
13
Ile to Phe at 601


V603F
G to T at 1939
13
Val to Phe at 603


T604I
C to T at 1943
13
Thr to Ile at 604


1949del84
deletion of 84 bp from 1949
13
deletion of 28 a.a. (Met607





to Gln634)


H609R
A to G at 1958
13
His to Arg at 609


L610S
T to C at 1961
13
Leu to Ser at 610


A613T
G to A at 1969
13
Ala to Thr at 613


D614Y
G to T at 1972
13
Asp to Tyr 614


D614G
A to G at 1973
13
Asp to Gly at 614


I618T
T to C at 1985
13
Ile to Thr at 618


L619S
T to C at 1988
13
Leu to Ser at 619


H620P
A to C at 1991
13
His to Pro at 620


H620Q
T to G at 1992
13
His to Gln at 620


G622D
G to A at 1997
13
Gly to Asp at 622





(oligospermia)


G628R(G->A)
G to A at 2014
13
Gly to Arg at 628


G628R(G->C)
G to C at 2014
13
Gly to Arg at 628


L633P
T to C at 2030
13
Leu to Pro at 633


Q634X
T to A at 2032
13
Gln to Stop at 634


L636P
T to C at 2039
13
Leu to Pro at 636


Q637X
C to T at 2041
13
Gln to Stop at 637


2043delG
deletion of G at 2043
13
Frameshift


2051delTT
deletion of TT from 2051
13
Frameshift


2055del9->A
deletion of 9 bp
13
Frameshift



CTCAAAACT to A at 2055


D648V
A to T at 2075
13
Asp to Val at 648


D651N
G to A at 2083
13
Asp to Asn at 651


E656X
T to G at 2098
13
Glu to Stop at 656


2108delA
deletion of A at 2108
13
Frameshift


2109del9->A
deletion of 9bp from 2109
13
Frameshift



and insertion of A


2113delA
deletion of A at 2113
13
Frameshift


2116delCTAA
deletion of CTAA at 2116
13
Frameshift


2118del4
deletion of AACT from 2118
13
Frameshift


E664X
G to T at 2122
13
Glu to Stop at 664


T665S
A to T at 2125
13
Thr to Ser at 665


2141insA
Insertion of A after 2141
13
Frameshift


2143delT
deletion of T at 2143
13
Frameshift


E672del
deletion of 3 bp between
13
deletion of Glu at 672



2145-2148


G673X
G to T at 2149
13
Gly to Stop at 673


W679X
G to A at 2168
13
Trp to stop at 679


2176insC
Insertion of C after 2176
13
Frameshift


K683R
A to G at 2180
13
Lys to Arg at 683


2183AA->G
A to G at 2183 and deletion
13
Frameshift



of A at 2184


2183delAA
deletion of AA at 2183
13
Frameshift


2184delA
deletion of A at 2184
13
frameshift


2184insG
inserion of G after 2184
13
Frameshift


2184insA
Insertion of A after 2184
13
Frameshift


2185insC
Insertion of C at 2185
13
Frameshift


Q685X
C to T at 2185
13
Gln to Stop at 685


E692X
G to T at 2206
13
Glu to Stop at 692


F693L(CTT)
T to C at 2209
13
Phe to Leu at 693


F693L(TTG)
T to G at 2211
13
Phe to Leu at 693


2215insG
Insertion of G at 2215
13
Frameshift


K698R
A to G 2225
13
Lys to Arg at 698


R709X
C to T at 2257
13
Arg to Stop at 709


K710X
A to T at 2260
13
Lys to Stop at 710


K716X
AA to GT at 2277 and 2278
13
Lys to Stop at 716


L719X
T to A at 2288
13
Leu to Stop at 719


Q720X
C to T at 2290
13
Gln to stop codon at 720


E725K
G to A at 2305
13
Glu to Lys at 725


2307insA
Insertion of A after 2307
13
Frameshift


E730X
G to T at 2320
13
Glu to Stop at 730


L732X
T to G at 2327
13
Leu to Stop at 732


2335delA
deletion of A at 2335
13
Frameshift


R735K
G to A at 2336
13
Arg to Lys at 735


2347delG
deletion of G at 2347
13
Frameshift


2372del8
deletion of 8 bp from 2372
13
Frameshift


P750L
C to T at 2381
13
Pro to Leu at 750


V754M
G to A at 2392
13
Val to Met at 754


T760M
C to T at 2411
13
Thr to Met at 760


R764X
C to T at 2422
13
Arg to Stop at 764


2423delG
deletion of G at 2423
13
Frameshift


R766M
G to T at 2429
13
Arg to Met at 766


2456delAC
deletion of AC at 2456
13
Frameshift


S776X
C to G at 2459
13
Ser to Stop at 776
















TABLE 13







CFTR mutations that may be detected in amplified product using


as the primer pair SEQ ID NO: 11 and 12.










Name
Nucleotide_change
Exon
Consequence





T908N
C to A at 2788
14b
Thr to Asn at 908


2789+2insA
insertion of A after
intron 14b
mRNA splicing defect



2789 + 2

(CAVD)


2789+3delG
deletion of G at
intron 14b
mRNA splicing defect



2789 + 3


2789+5G->A
G to A at 2789 + 5
intron 14b
mRNA splicing defect
















TABLE 14







CFTR mutations that may be detected in amplified product using


as the primer pair SEQ ID NO: 25 and 26.










Name
Nucleotide_change
Exon
Consequence





3100insA
Insertion of A after
16
Frameshift



3100


I991V
A to G at 3103
16
Ile to Val at 991


D993Y
G to T at 3109
16
Asp to Tyr at 993


F994C
T to G at 3113
16
Phe to Cys at 994


3120G->A
G to A at 3120
16
mRNA splicing defect


3120+1G->A
G to A at 3120 + 1
intron 16
mRNA splicing defect
















TABLE 15







CFTR mutations that may be detected in amplified product using as the primer


pair SEQ ID NO: 29 and 30.










Name
Nucleotide_change
Exon
Consequence





3601-20T->C
T to C at 3601-20
intron 18
mRNA splicing mutant


3601-17T->C
T to C at 3601-17
intron 18
mRNA splicing defect


3601-2A->G
A to G at 3601-2
intron 18
mRNA splicing defect


R1158X
C to T at 3604
19
Arg to Stop at 1158


S1159P
T to C at 3607
19
Ser to Pro at 115p


S1159F
C to T at 3608
19
Ser to Phe at 1159


R1162X
C to T at 3616
19
Arg to Stop at 1162


3622insT
Insertion of T after 3622
19
Frameshift


D1168G
A to G at 3635
19
Asp to Gly at 1168


3659delC
deletion of C at 3659
19
Frameshift


K1177X
A to T at 3661
19
Lys to Stp at 3661





(premature termination)


K1177R
A to G at 3662
19
Lys to Arg at 1177


3662delA
deletion of A at 3662
19
Frameshift


3667del4
deletion of 4 bp from 3667
19
Frameshift


3667ins4
insertion of TCAA after 3667
19
Frameshift


3670delA
deletion of A at 3670
19
Frameshift


Y1182X
C to G at 3678
19
Tyr to Stop at 1182


Q1186X
C to T at 3688
19
Gln to Stop codon at 1186


3696G/A
G to A at 3696
19
No change to Ser at 1188


V1190P
T to A at 3701
19
Val to Pro at 1190


S1196T
C or Q at 3719
19
Ser-Top at 1196


S1196X
C to G at 3719
19
Ser to Stop at 1196


3724delG
deletion of G at 3724
19
Frameshift


3732delA
deletion of A at 3732 and A
19
frameshift and Lys to Glu at



to G at 3730

1200


3737delA
deletion of A at 3737
19
Frameshift


W1204X
G to A at 3743
19
Trp to Stop at 1204


S1206X
C to G at 3749
19
Ser to Stop at 1206


3750delAG
deletion of AG from 3750
19
Frameshift


3755delG
deletion of G between 3751
19
Frameshift



and 3755


M1210I
G to A at 3762
19
Met to Ile at 1210


V1212I
G to A at 3766
19
Val to Ile at 1212
















TABLE 16







CFTR mutations that may be detected in amplified product using


as the primer pair SEQ ID NO: 5 and 6.










Name
Nucleotide_change
Exon
Consequence





3849+10kbC->T
C to T in a 6.2 kb EcoRI
intron 19
creation of



fragment 10 kb from 19

splice acceptor





site
















TABLE 17







CFTR mutations that may be detected in amplified product using


as the primer pair SEQ ID NO: 17 and 18.










Name
Nucleotide_change
Exon
Consequence





T1252P
A to C at 3886
20
Thr to Pro at 1252


L1254X
T to G at 3893
20
Leu to Stop at 1254


S1255P
T to C at 3895
20
Ser to Pro at 1255


S1255L
C to T at 3896
20
Ser to Leu at 1255


S1255X
C to A at 3896 and A to G at
20
Ser to Stop at 1255 and Ile to



3739 in exon 19

Val at 1203


3898insC
Insertion of C after 3898
20
Frameshift


F1257L
T to G at 3903
20
Phe to Leu at 1257


3905insT
Insertion of T after 3905
20
Frameshift


3906insG
Insertion of G after 3906
20
Frameshift


ΔL1260
deletion of ACT from either
20
deletion of Leu at 1260 or



3909 or 3912

1261


3922del10->C
deletion of 10 bp from 3922
20
deletion of Glu1264 to



and replacement with 3921

Glu1266


I1269N
T to A at 3938
20
Ile to Asn at 1269


D1270N
G to A at 3940
20
Asp to Asn at 1270


3944delGT
deletion of GT from 3944
20
Frameshift


W1274X
G to A at 3954
20
Trp to Stop at 1274


Q1281X
C to T at 3973
20
Gln to Stop at 1281


W1282R
T to C at 3976
20
Trp to Arg at 1282


W1282G
T to G at 3976
20
Trp to Gly at 1282


W1282X
G to A at 3978
20
Trp to Stop at 1282


W1282C
G to T at 3978
20
Trp to Cys at 1282


R1283M
G to T at 3980
20
Arg to Met at 1283


R1283K
G to A at 3980
20
Arg to Lys at 1283


F1286S
T to C at 3989
20
Phe to Ser at 1286
















TABLE 18







CFTR mutations that may be detected in amplified product using


as the primer pair SEQ ID NO: 13 and 14.










Name
Nucleotide_change
Exon
Consequence





T1299I
C to T at 4028
21
Thr to Ile at 1299


F1300L
T to C at 4030
21
Phe to Leu at 1300


N1303H
A to C at 4039
21
Asn to His at 1303


N1303I
A to T at 4040
21
Asn to Ile at 1303


4040delA
deletion of A at 4040
21
Frameshift


N1303K
C to G at 4041
21
Asn to Lys at 1303


D1305E
T to A at 4047
21
Asp to Glu at 1305


4048insCC
insertion of CC after 4048
21
Frameshift


Y1307X
T to A at 4053
21
Tyr to Stop at 1307


E1308X
G to T at 4054
21
Glu to Stop at 1308









CF mutations including those known under symbols: 2789+5G>A; 711+1G>T; W1282X; 3120+1G>A; d1507; dF508; (F508C, 1507V, 1506V); N1303K; G542X, G551D, R553X, R560T, 1717-1G>A: R334W, R347P, 1078delT; R117H, 1148T, 621+1G>T; G85E; R1162X, 3659delC; 2184delA; A455E, (5T, 7T, 9T); 3849+10 kbC>T; and 1898+1G>A, are described in U.S. patent application Ser. No. 396,894, tiled Apr. 22, 1989, U.S. patent application Ser. No. 399,945, filed Aug. 29, 1989, U.S. patent application Ser. No. 401,609 filed Aug. 31, 1989. and U.S. Pat. Nos. 6,001,588 and 5,981,178, which are hereby incorporated by reference in their entirety. Any and all of these mutations can be detected using nucleic acid amplified with the invention primers as described herein.
CF mutations in the amplified nucleic acid may be identified in any of a variety of ways well known to those of ordinary skill in the art. For example, if an amplification product is of a characteristic size, the product may be detected by examination of an electrophoretic gel for a band at a precise location. In another embodiment, probe molecules that hybridize to the mutant or wildtype CF sequences can be used for detecting such sequences in the amplified product by solution phase or, more preferably, solid phase hybridization. Solid phase hybridization can be achieved, for example, by attaching the CF probes to a microchip. Probes for detecting CF mutant sequences are well known in the art. See Wall et al. “A 31-mutation assay for cystic fibrosis testing in the clinical molecular diagnostics laboratory,” Human Mutation, 1995; 5(4):333-8, which specifies probes for CF mutations ΔF508 (exon 1), G542X (exon 11), G551D (exon 11), R117H (exon 4), W1282X (exon 20), N1303K (exon 21), 3905insT (exon 20), 3849+10 Kb (intron 19), G85E (exon 3), R334W (exon 7), A455E (exon 9), 1898+1 (exon 12), 2184delA (exon 13), 711+1 (exon 5), 2789+5 (exon 14b), Y1092x exon 17b), ΔI507 (exon 10), S549R(T-G) (exon 11), 621+1 (exon 4), R1162X (exon 19), 1717-1 (exon 11), 3659delC (exon 19), R560T (exon 11), 3849+4(A-G) (exon 19), Y122X (exon 4), R553X (exon 11), R347P (exon 7), R347H (exon 7), Q493X (exon 10), V520F (exon 10), and S549N (exon 11).
CF probes for detecting mutations as described herein may be attached to a solid phase in the form of an array as is well known in the art (see, U.S. Pat. Nos. 6,403,320 and 6,406,844). For example, the full complement of 24 probes for CF mutations with additional control probes (30 in total) can be conjugated to a silicon chip essentially as described by Jenison et al Biosens Bioelectron, 16(9-12):757-63 (2001) (see also U.S. Pat. Nos. 6,355,429 and 5,955,377). Amplicons that hybridized to particular probes on the chip can be identified by transformation into molecular thin films. This can be achieved by contacting the chip with an anti-biotin antibody or streptavidin conjugated to an enzyme such as horseradish peroxidase. Following binding of the antibody (or streptavidin)-enzyme conjugate to the chip, and washing away excess unbound conjugate, a substrate can be added such as tetramethylbenzidine (TMB) {3,3′,5,5′Tetramethylbenzidine} to achieve localized deposition (at the site of bound antibody) of a chemical precipitate as a thin film on the surface of the chip. Other enzyme/substrate systems that can be used are well known in the art and include, for example, the enzyme alkaline phosphatase and 5-bromo-4-chloro-3-indolyl phosphate as the substrate. The presence of deposited substrate on the chip at the locations in the array where probes are attached can be read by an optical scanner. U.S. Pat. Nos. 6,355,429 and 5,955,377, which are hereby incorporated by reference in their entirety including all charts and drawings, describe preferred devices for performing the methods of the present invention and their preparation, and describes methods for using them.
The binding of amplified nucleic acid to the probes on the solid phase following hybridization may be measured by methods well known in the art including, for example, optical detection methods described in U.S. Pat. No. 6,355,429. In preferred embodiments, an array platform (see, e.g., U.S. Pat. No. 6,288,220) can be used to perform the methods of the present invention, so that multiple mutant DNA sequences can be screened simultaneously. The array is preferably made of silicon, but can be other substances such as glass, metals, or other suitable material, to which one or more capture probes are attached. In preferred embodiments, at least one capture probe for each possible amplified product is attached to an array. Preferably an array contains 10, more preferably 20, even more preferably 30, and most preferably at least 60 different capture probes covalently attached to the array, each capture probe hybridizing to a different CF mutant sequence. Nucleic acid probes useful as positive and negative controls also may be included on the solid phase or used as controls for solution phase hybridization.
Another approach, variously referred to as PCR amplification of specific allele (PASA) (Sarkar et al., 1990 Anal. Biochem. 186:64-68), allele-specific amplification (ASA) (Okayama, et al., 1989 J. Lab. Clin. Med. 114:105-113), allele-specific PCR (ASPCR) (Wu, et al. 1989 Proc. Natl. Acad. Sci. USA. 86:2757-2760), and amplification-refractory mutation system (ARMS) (Newton, et al., 1989 Nucleic Acids Res. 17:2503-2516). The method is applicable for single base substitutions as well as micro deletions/insertions. In general, two complementary reactions are used. One contains a primer specific for the normal allele and the other reaction contains a primer for the mutant allele (both have a common 2nd primer). One PCR primer perfectly matches one allelic variant of the target, but is mismatched to the other. The mismatch is located at/near the 3′ end of the primer leading to preferential amplification of the perfectly matched allele. Genotyping is based on whether there is amplification in one or in both reactions. A hand in the normal reaction only indicates a normal allele. A hand in the mutant reaction only indicates a mutant allele. Bands in both reactions indicate a heterozygote. As used herein, this approach will be referred to as “allele specific amplification.”
In yet another approach, restriction fragment length polymorphism (RFLP), which refers to the digestion pattern of various restriction enzymes applied to DNA. RFLP analysis can be applied to PCR amplified DNA to identify CF mutations as disclosed herein.
In still another approach, wildtype or mutant CF sequence in amplified DNA may be detected by direct sequence analysis of the amplified products. A variety of methods can be used for direct sequence analysis as is well known in the art. See, e.g., The PCR Technique: DNA Sequencing (eds. James Ellingboe and Ulf Gyllensten) Biotechniques Press, 1992; see also “SCAIP” (single condition amplification/internal primer) sequencing, by Flanigan et al. Am J Hum Genet. 2003 April; 72(4):931-9. Epub 2003 Mar. 11. Direct sequencing of CF mutations is also described in Strom et al., 2003 Genetics in Medicine 5(1):9-14.
In yet another approach for detecting wildtype or mutant CF sequences in amplified DNA is single nucleotide primer extension or “SNuPE.” SNuPE can be performed as described in U.S. Pat. No. 5,888,819 to Goelet et al., U.S. Pat. No. 5,846,710 to Bajaj, Piggee, C. et al. Journal of Chromatography A 781 (1997), p. 367-375 (“Capillary Electrophoresis for the Detection of Known Point Mutations by Single-Nucleotide Primer Extension and Laser-Induced Fluorescence Detection”); Hoogendoorn, B. et al., Human Genetics (1999) 104:89-93, (“Genotyping Single Nucleotide Polymorphism by Primer Extension and High Performance Liquid Chromatography”); and U.S. Pat. No. 5,885,775 to Haff et al. (analysis of single nucleotide polymorphism analysis by mass spectrometry). In SNuPE, one may use as primers such as those specified in Table 17.
Another method for detecting CF mutations include the Luminex xMAP system which has been adapted for cystic fibrosis mutation detection by TM Bioscience and is sold commercially as a universal bead array (Tag-ItTM)
Still another approach for detecting wildtype or mutant CF sequences in amplified DNA is oligonucleotide ligation assay or “OLA” or “OL”. The OLA uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules. One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected. See e.g., Nickerson et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:8923-8927, Landegren, U. et al. (1988) Science 241:1077-1080 and U.S. Pat. No. 4,998,617.
These above approaches for detecting wildtype or mutant CF sequence in the amplified nucleic acid is not meant to be limiting, and those of skill in the art will understand that numerous methods are known for determining the presence or absence of a particular nucleic acid amplification product.
In another aspect the present invention provides kits for one of the methods described herein. The kit optionally contain buffers, enzymes, and reagents for amplifying the CFTR nucleic acid via primer-directed amplification. The kit also may include one or more devices for detecting the presence or absence of particular mutant CF sequences in the amplified nucleic acid. Such devices may include one or more probes that hybridize to a mutant CF nucleic acid sequence, which may be attached to a bio-chip device, such as any of those described in U.S. Pat. No. 6,355,429. The bio-chip device optionally has at least one capture probe attached to a surface on the bio-chip that hybridizes to a mutant CF sequence. In preferred embodiments the bio-chip contains multiple probes, and most preferably contains at least one probe for a mutant CF sequence which, if present, would be amplified by a set of flanking primers. For example, if five pairs of flanking primers are used for amplification, the device would contain at least one CF mutant probe for each amplified product, or at least five probes. The kit also preferably contains instructions for using the components of the kit.
The following examples serve to illustrate the present invention. These examples are in no way intended to limit the scope of the invention
EXAMPLES
Example 1
Sample Collection and Preparation
Whole Blood: 5 cc of whole blood is collected in a lavender-top (EDTA) tube or yellow-top (ACD) tube. Green-top (Na Heparin) tubes are acceptable but less desirable. DNA is extracted from blood. 100 ng or more DNA is prepared in TE or sterile water.
Amniotic Fluid: 10-15 cc of Amniotic Fluid is collected in a sterile plastic container.
Cultured Cells: Two T-25 culture flasks with 80-100% confluent growth may be used.
Chorionic 10-20 mg of Chorionic Villi are collected in a sterile container. 2-3 mL of sterile saline or tissue culture medium is added.
Transport: Whole Blood, Amniotic Fluid, Cultured Cells and Chorionic Villi can be shipped at room temperature (18°-26° C.). Amniotic Fluid, Cultured Cells or Chorionic Villi preferably is used without refrigeration or freezing. Whole Blood and Extracted DNA can be shipped at 2°-10° C.
Storage: Whole Blood, Amniotic Fluid and Extracted DNA are stored at 2°-10° C., Cultured Cells and Chorionic Villi are stored at room temperature (18°-26° C.).
Stability: Whole Blood is generally stable for 8 days at room temperature (18°-26° C.) or 8 days refrigerated at 2°-10° C. Amniotic Fluid, Cultured Cells, and Chorionic Villi are generally processed to obtain DNA within 24 hours of receipt. Extracted DNA is stable for at least 1 year at 2°-10° C.
Example 2
Amplification from DNA
Polymerase chain reaction (PCR) primer pairs were designed using the CFTR gene sequences in EMBL/Genbank (Accession Nos. M55106-M55131). Each PCR primer for the 32 separate PCR reactions contains either an M13 forward linker sequence or an M13 reverse linker sequence as appropriate to allow universal sequence reaction priming. Individual PCR reactions are performed in 96-well microtiter plates under the same conditions for each amplicon. Subsequently, the PCR products are purified with the Millipore MontageTM PCR96 Cleanup kit (Millipore, Bedford, Mass.) on a Beckman BioMek 2,000 biorobot. Further details are provided in Strom et al., 2003 Genetics in Medicine 5(1):9-14.
In general, individual amplifications are prepared in a volume of 13.5 which is added to the 96 well microtiter plates. Each amplification volume contained 2 μl of the nucleic acid sample (generally 10-100 ng of DNA), 11.5 μl of PCR-Enzyme Mix (PCR-Enzyme mix stock is prepared with 11.3 μl master mix, 0.25 μl MgCl2 (from 25 mM stock), and 0.2 μl of FasStar Taq (source for last two reagents was Roche Applied science, Cat. No. 2 032 937). Master mix contained primers, Roche PCR buffer with MgCl2, Roche GC rich solution (cat. No. 2 032 937), bovine serum albumin (BSA) (New England BioLabs, Cat no. B9001B), and NTPs (Amersham Biosciences, Cat no. 27-2032-01).
The final concentration in the PCR for MgCl2 was 2.859 mM, for BSA is 0.725 μg, and for each dNTP is 0.362 mM. Primer final concentrations varied from about 0.29 μM to about 0.036 μM
PCR is conducted using the following temperature profile: step 1: 96° C. for 15 minutes; step 2: 94° C. for 15 seconds; step 3: decrease at 0.5° C./second to 56° C.; step 4: 56° C. for 20 seconds; step 5: increase at 0.3° C./second to 72° C., step 6: 72° C. for 30 seconds; step 7: increase 0.5° C. up to 94° C.; step 8: repeat steps 2 to 7 thirty three times; step 9: 72° C. for 5 minutes; step 10: 4° C. hold (to stop the reaction).
Example 3
Detection of CF Mutations
The purified PCR products are diluted to approximately 10 ng/μL and cycle sequencing reactions are performed with an ABI Prism Big DyeTM Terminator v3.0 cycle sequencing reaction kit (Applied Biosystems, Foster City, Calif.) according to the manufacturer's protocol. The DNA primers used for the sequencing reaction are M13 forward and reverse primers as appropriate. Big DyeTM Terminator reaction products are purified by the Millipore MontageTM Seq96 Sequencing Reaction Cleanup kit on a biorobot and analyzed on an ABI Prism 3100 Genetic Analyzer. Sequences obtained are examined for the presence of mutations by using ABI SeqScape v1.1 software. Both strands of DNA are sequenced.
All PCR reactions, purifications, and cycle sequencing reactions are performed in 96-well microtiter plates using biorobots to avoid errors introduced by manual setups. Loading of samples onto the capillary sequencer is also automated. One plate is sufficient to perform the entire sequencing reaction for a single patient. Theoretically, if all reactions were successful, the entire sequences for a single patient could be obtained in 24-48 hours after receipt of blood. In practice, however, one or more reactions must be repeated because of frequent polymorphisms in intron 8 and 6a and failed reactions.
The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.
The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

## Claims

1. A method of determining the cystic fibrosis status of a human comprising:
(a) determining the presence or absence of the c.3297C>A mutation, which results in F1099L/p.Phe1099Leu, in both alleles of the cystic fibrosis transmembrane regulatory (CFTR) gene in a nucleic acid sample obtained from the human, and
(b) identifying the human
(i) as having cystic fibrosis or being predisposed to cystic fibrosis when the human is homozygous for the c.3297C>A mutation in the CFTR gene, or
(ii) as being a cystic fibrosis carrier when the human is heterozygous for the c.3297C>A mutation in the CFTR gene, or
(iii) as having no predisposition or carrier status caused by the c.1352G>T mutation when the c.3297C>A mutation is absent from both alleles of the CFTR gene.

2. A method of determining if a human is predisposed to cystic fibrosis comprising:
(a) determining the presence or absence of the c.3297C>A mutation, which results in F1099L/p.Phe1099Leu. in both alleles of the cystic fibrosis transmembrane regulatory (CFTR) gene in a nucleic acid sample obtained from the human, and
(b) identifying said human
(i) as being predisposed to cystic fibrosis when the human is homozygous for the c.3297C>A mutation in the CFTR gene, or
(ii) as having no predisposition to cystic fibrosis when the human is heterozygous for the c.3297C>A mutation in the CFTR gene or the c.3297C>A mutation is absent from both alleles of the CFTR gene.

3. A method of identifying a human as having an increased likelihood of having an offspring afflicted with cystic fibrosis, comprising
(a) determining the presence or absence of the c.3297C>A mutation, which results in F1099L/p.Phe1099Leu, in both alleles of the CFTR gene in a nucleic acid sample obtained from the human, and
(b) identifying said human as having an increased likelihood of having an offspring afflicted with cystic fibrosis when said c.3297C>A mutation is present in at least one allele.

4. The method of any one of claims 1-3 wherein genomic DNA is tested for the presence of said mutation.

5. The method of any one of claims 1-3 wherein a cDNA copy of the CFTR coding sequence is tested for the presence of said mutation.

6. The method of any one of claims 1-3 wherein the presence of said mutation is determined by nucleic acid sequencing.

7. The method of any one of claims 1-3 wherein the presence of said mutation is determined by restriction fragment length polymorphism analysis following treatment of CFTR gene sequence with at least one endonuclease restriction enzyme.

8. The method of any one of claims 1-3 wherein the presence of said mutation is determined by allele specific amplification.

9. The method of any one of claims 1-3 wherein the presence of said mutation is determined by primer extension.

10. The method of any one of claims 1-3 wherein the presence of said mutation is determined by oligonucleotide ligation.

11. The method of any one of claims 1-3 wherein the presence of said mutation is determined by hybridization with a detectably labeled probe.