US5068176A
Expired / Lapsed
US
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Method for the simultaneous determination of dna sequence variations at a large number of sites, and a kit suitable therefor
Assignee: NEDERLANDSE ORGANISATIE VOOR TOEGEPASTNATUURWETENSCHAPPELIJK ONDERZOEK TNO A CORP OF NETHERLANDS
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Filing: 1989-05-01
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Granted: 1991-11-26
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Expires: 2009-05-01
Abstract
Abstract This invention relates to a method of simultaneously determining DNA sequence variations on a large number of loci, and a kit suitable therefor. The method comprises an electrophoretic separation of restriction fragments in two dimensions on the basis of two independent criterions, namely, length and base pair sequence, and a detection of separated fragments by means of a labelled probe comprising one or more units of minisatellite sequences. Preferred probes are GC-rich minisatellite core sequences.
Inventors
- 👤 Jan Vijg
- 👤 Andreas G. Uitterlinden
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 17:56
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; C12Q1/6813; C12Q1/6827; C12Q1/683; C12Q2600/00; C12Q2600/156; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S436/00; Y10S436/807; Y10S436/808
Claims Analysis
Total Claims: 10
Claim 1
Independent Claim
130 words
A method of detecting genetic variation by fragmenting double-stranded DNA by means of one or more restriction enzymes, separating the resulting fragments by electrophoresis, transferring the resulting separation pattern to a membrane filter, hybridizing thereon under DNA-denaturing conditions with a labelled DNA or RNA probe, and visualizing the label, characterized in that the electrophoretic separation of the DNA fragments generated is carried out in two dimensions on the basis of two independent criteria, the fragments being separated in one dimension on the basis of their length and being separated in the other dimension on the basis of their base pair sequence, and in that the probe used in the hybridization analysis is a labelled DNA or RNA molecule containing one or more cores of minisatellite sequences or other GC-rich repeats.
Claim 2
Independent Claim
130 words
A method of detecting genetic variation by fragmenting double-stranded DNA by means of one or more restriction enzymes, separating the resulting fragments by electrophoresis, transferring the resulting separation pattern to a membrane filter, hybridizing thereon under DNA-denaturing conditions with a labelled DNA or RNA probe, and visualizing the label, characterized in that the electrophoretic separation of the DNA fragments generated is carried out in two dimensions on the basis of two independent criteria, the fragments being separated in one dimension on the basis of their length and being separated in the other dimension on the basis of their base pair sequence, and in that the probe used in the hybridization analysis is a labelled DNA or RNA molecule containing one or more cores of minisatellite sequences or other GC-rich repeats.
Claim 3
Dependent Claim
63 words
A method as claimed in claim 1, characterized in that the DNA fragments generated are first separated on the basis of their length in a neutral polyacrylamide gel, and subsequently, in the second dimension, are separated on the basis of their base pair sequence in a polyacrylamide gel having an increasing concentration gradient of a DNA denaturant parallel to the direction of electrophoresis.
Claim 4
Dependent Claim
53 words
A method as claimed in claim 2, characterized in that the electrophoretic separation on the basis of length is carried out at a temperature of between 45° and 55° C, and the electrophoretic separation on the basis of base pair sequence is carried out at a temperature of between 55° and 65° C.
Claim 5
Dependent Claim
44 words
A method as claimed in claim 2, characterized in that the separation pattern obtained after the two-dimensional electrophoresis is transferred to a membrane filter by electroblotting, and that prior to said transfer the separation pattern is fragmented by irradiation and further denatured by heating.
Claim 6
Dependent Claim
20 words
A method as claimed in claim 1, characterized by using as the membrane filter a nylon membrane or comparable material.
Claim 7
Dependent Claim
37 words
A method as claimed in claim 1, characterized by using as a probe a DNA molecule containing one or more GC-rich cores for repeat sequences, such as minisatellites or simple sequences or a mixture of locus-specific probes.
Claim 8
Dependent Claim
45 words
A method as claimed in claim 1, characterized in that the DNA fragments generated are derived from specially constructed cDNA libraries, in which GC-rich sequence have been attached to cDNA molecules prepared from complex RNA populations (such as total RNA from organs, tissues or cells).
Claim 9
Dependent Claim
27 words
A method as claimed in claim 1, characterized by using a probe labelled with a radioactive isotope, for example 32 P or non-radioactive label, such as biotin.
Claim 10
Dependent Claim
57 words
A kit for use in the method as claimed in claim 1, comprising a labelled probe containing one or more cores of minisatellite sequences or other GC-rich repeats and material for two-dimensional electrophoresis containing a geld with concentration gradient of a DNA denaturant, suitable for separation of DNA fragments on the basis of their basie pair sequence.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1994-11-25 | 1995-05-27 | 1995-11-26 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1998-11-25 | 1999-05-27 | 1999-11-26 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2002-11-25 | 2003-05-27 | 2003-11-26 | $7700.00 | Window Closed / Lapsed |