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US4395486A
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Method for the direct analysis of sickle cell anemia
Assignee: Augusta University Research Institute Inc
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Filing: 1981-08-19
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Granted: 1983-07-26
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Expires: 2001-08-19
Abstract
Abstract A direct diagnosis of sickle cell anemia by a restriction endonuclease assay with an enzyme which recognizes the nucleotide base sequence CTNAG (where N is any base), such as Dde I, for the sickle cell allele (β s gene) through molecular hybridization. Following enzyme cleavage, the resulting DNA restriction fragments are separated by molecular weight and transferred to filter paper. A probe is utilized for hybridization that is complementary to the 5' end of the β globin gene. The banding pattern of individuals with normal hemoglobin shows two bands (approximately 175 bp and 201 bp), sickle cell trait individuals exhibit an additional band (approximately 376 bp) and individuals with sickle cell anemia show the band at approximately 376 bp with a concommitant loss of the band at approximately 175 bp. Prenatal and postnatal diagnosis of sickle cell anemia is possible with the present method.
Inventors
- 👤 Lois B. Wilson
- 👤 John T. Wilson
- 👤 Robert F. Geever
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 22:58
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; C12Q1/6883; C12N; C12N15/00; C12Q1/6813; C12Q1/6827; C12Q1/683; C12Q2600/00; C12Q2600/156
Claims Analysis
Total Claims: 30
Claim 1
Independent Claim
102 words
A method for the direct analysis of sickle cell anemia in the human β gene, comprising the steps of: (a) obtaining an effective amount of amniotic fluid cells from a human; (b) isolating DNA from said cells; (c) digesting said DNA with restriction enzyme Dde I to form DNA fragments; (d) separating said DNA fragments according to their respective molecular weights to form a pattern for said fragments on filter paper; and (e) detecting the absence of an approximate 175 base pair fragment and the presence of an approximate 376 base pair fragment on said pattern, thereby indicating the sickle cell genotype.
Claim 2
Independent Claim
102 words
A method for the direct analysis of sickle cell anemia in the human β gene, comprising the steps of: (a) obtaining an effective amount of amniotic fluid cells from a human; (b) isolating DNA from said cells; (c) digesting said DNA with restriction enzyme Dde I to form DNA fragments; (d) separating said DNA fragments according to their respective molecular weights to form a pattern for said fragments on filter paper; and (e) detecting the absence of an approximate 175 base pair fragment and the presence of an approximate 376 base pair fragment on said pattern, thereby indicating the sickle cell genotype.
Claim 3
Dependent Claim
18 words
A method as claimed in claim 1 wherein said separating step includes gel electrophoresis of said DNA fragments.
Claim 4
Dependent Claim
13 words
A method as claimed in claim 2 wherein said gels are polyacrylamide gels.
Claim 5
Dependent Claim
21 words
A method as claimed in claim 2 further including the step of electrophoretically transferring said DNA fragments to DBM filter paper.
Claim 6
Dependent Claim
22 words
A method as claimed in claim 1 wherein said separating step includes isolating said fragments by the use of reversed phase chromatography.
Claim 7
Dependent Claim
37 words
A method as claimed in claim 1 wherein said separating step includes directing said fragments through a high pressure reversed phase chromatographic column (RPC-5) to obtain separated fragments and transferring said separated fragments to said filter paper.
Claim 8
Dependent Claim
20 words
A method as claimed in claim 1 wherein said detecting step includes hybridizing said separated fragments with a DNA probe.
Claim 9
Dependent Claim
29 words
A method as claimed in claim 7 wherein said probe comprises recombinant human DNA having the β globin genomic sequence included therein, said sequence labeled with a radioactive nucleotide.
Claim 10
Dependent Claim
29 words
A method as claimed in claim 7 wherein said probe comprises recombinant human DNA having the δ globin genomic sequence included therein, said sequence labeled with a protein-coupled nucleotide.
Claim 11
Dependent Claim
30 words
A method as claimed in claim 7 wherein said probe comprises recombinant human DNA having the β globin genomic sequence included therein, said sequence labeled with a biotin coupled nucleotide.
Claim 12
Dependent Claim
24 words
A method as claimed in claim 8 and further including the step of blot hybridizing said probe with said fragments on said filter paper.
Claim 13
Dependent Claim
19 words
A method as claimed in claim 11 including the step of preparing an autoradiogram of the hybridized filter paper.
Claim 14
Dependent Claim
28 words
A method as claimed in either claim 9 or claim 10 and further including the step of blot hybridizing said probe with said fragments on said filter paper.
Claim 15
Dependent Claim
25 words
A method as claimed in claim 13 and further including the step of coating said filter paper with fluorescent antibodies and illuminating with ultraviolet light.
Claim 16
Independent Claim
78 words
A method for the direct analysis of sickle cell anemia in genomic DNA, comprising the steps of: (a) digesting said DNA with restriction endonuclease Dde I to generate fragments; (b) resolving said fragments according to their respective molecular weights; (c) transferring said fragments to filter paper; and (d) visualizing said fragments on said filter paper to detect an approximate 376 base pair fragment and the absence of an approximate 175 base pair fragment, thereby determining sickle cell anemia.
Claim 17
Independent Claim
78 words
A method for the direct analysis of sickle cell anemia in genomic DNA, comprising the steps of: (a) digesting said DNA with restriction endonuclease Dde I to generate fragments; (b) resolving said fragments according to their respective molecular weights; (c) transferring said fragments to filter paper; and (d) visualizing said fragments on said filter paper to detect an approximate 376 base pair fragment and the absence of an approximate 175 base pair fragment, thereby determining sickle cell anemia.
Claim 18
Dependent Claim
35 words
A method as claimed in claim 15 wherein the steps prior to said digesting step include obtaining an effective amount of amniotic fluid cells from a human and isolating said DNA from said fluid cells.
Claim 19
Dependent Claim
18 words
A method as claimed in claim 15 wherein said resolving step includes separating said fragments on polyacrylamide gels.
Claim 20
Dependent Claim
26 words
A method as claimed in claim 15 wherein said resolving step includes the step of directing said fragments through a high pressure reversed phase chromatographic column.
Claim 21
Dependent Claim
23 words
A method as claimed in claim 15 wherein said visualizing step comprises hybridizing said fragments on said filter paper with a DNA probe.
Claim 22
Dependent Claim
28 words
A method as claimed in claim 19 wherein said probe comprises recombinant human DNA having included therein a sequence comprising the 5' end of the β globin gene.
Claim 23
Dependent Claim
14 words
A method as claimed in claim 20 wherein said sequence includes a radioactive nucleotide.
Claim 24
Dependent Claim
21 words
A method as claimed in claim 21 and further including the step of preparing an autoradiogram of the hybridized filter paper.
Claim 25
Dependent Claim
14 words
A method as claimed in claim 20 wherein said sequence includes a protein-coupled nucleotide.
Claim 26
Dependent Claim
14 words
A method as claimed in claim 20 wherein said sequence includes a biotin-coupled nucleotide.
Claim 27
Dependent Claim
32 words
A method as claimed in either claim 23 or claim 24 and further including the steps of binding a fluorescent antibody to said nucleotide and illuminating said filter paper with ultraviolet light.
Claim 28
Independent Claim
94 words
A method for the direct analysis of sickle cell anemia in genomic DNA, comprising the steps of: (a) digesting said DNA with a restriction enzyme which recognizes the nucleotide base sequence CTNAG wherein N is any base to produce fragments; (b) separating said fragments according to their respective molecular weights; (c) hybridizing said fragments with a DNA probe; and (d) visualizing said pattern to determine if the recognition sequence of said restriction enzyme is created or eliminated by the sickle cell allele, the elimination of said sequence indicating the presence of sickle cell genotype.
Claim 29
Independent Claim
94 words
A method for the direct analysis of sickle cell anemia in genomic DNA, comprising the steps of: (a) digesting said DNA with a restriction enzyme which recognizes the nucleotide base sequence CTNAG wherein N is any base to produce fragments; (b) separating said fragments according to their respective molecular weights; (c) hybridizing said fragments with a DNA probe; and (d) visualizing said pattern to determine if the recognition sequence of said restriction enzyme is created or eliminated by the sickle cell allele, the elimination of said sequence indicating the presence of sickle cell genotype.
Claim 30
Dependent Claim
14 words
A method as claimed in claim 26 wherein said restriction enzyme is Dde I.
References Cited (Backward Citations)
US4298685A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1986-07-25 | 1987-01-24 | 1987-07-26 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-07-25 | 1991-01-24 | 1991-07-26 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-07-25 | 1995-01-24 | 1995-07-26 | $7700.00 | Window Closed / Lapsed |