EP0157280A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
DNA sequencing
Assignee: University of Nebraska–Lincoln
•
Filing: 1985-03-19
•
Granted: —
•
Expires: 2005-03-19
Abstract
Abstract To sequence long strands of DNA, cloned strands having lengths longer than 100 bases are, in one embodiment, marked on one end with biotin. These strands are divided into 4 aliquots and each aliquot: (1) is uniquely chemically treated to randomly terminate the strands at the non-biotinylated end at a selected type of base; and (2) is moved continuously by electrophoresis through a different one of four identical channels. In the one embodiment, the strands are randomly terminated at a selected base type and they are moved into avidin, which due to high affinity, combines with the biotin marked ends of shorter strands before the longer strands are fully resolved in the gel. The avidin is marked with fluorescein, the strands are scanned and the signals are decoded. In another embodiment, the strands are synthesized, with termination at a selected base type and marked either by the above method or by ethidium bromide.
Inventors
- 👤 John Albert Brumbaugh
- 👤 Lyle Richard Middendorf
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-26 07:24
CPC Classifications
G; G01; G01N; G01N27/00; G01N27/26; G01N27/416; G01N27/447; G01N27/44704; G01N27/44717; G01N27/44721; G01N27/44726; C; C07; C07D; C07D209/00; C07D209/56; C07D209/58; C07D209/60; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6869; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/6428; Y; Y10; Y10S; Y10S435/00; Y10S435/803
Claims Analysis
Total Claims: 8
Claim 1
Independent Claim
129 words
Apparatus for sequencing DNA (e.g. FIG. 1 or FIG. 2) using a gel (FIG. 3, 27) or capillary electrophoresis apparatus (26A); having a first separating apparatus (26A, 26G, 26C, 26T in FIG. 1) with at least four identical channels (26A, 26G, 26C, 26T of FIG. 1 or A26A, A26C, A26G, A26T of FIG. 2) and at least four separate channel-introducing sections (20A, 20G, 20C, 20T) each adapted to receive DNA fragments terminated at different ones of the four nucleic acid molecules, A, G, C and T respectively; characterized by advancing aparatus (47A, 53) for moving fragments from the end (31A) of said separating apparatus prior to the resolution of the last to be introduced bands while maintaining four separate groups of DNA fragments in accordance with the terminating base.
Claim 2
Dependent Claim
129 words
Apparatus for sequencing DNA (e.g. FIG. 1 or FIG. 2) using a gel (FIG. 3, 27) or capillary electrophoresis apparatus (26A); having a first separating apparatus (26A, 26G, 26C, 26T in FIG. 1) with at least four identical channels (26A, 26G, 26C, 26T of FIG. 1 or A26A, A26C, A26G, A26T of FIG. 2) and at least four separate channel-introducing sections (20A, 20G, 20C, 20T) each adapted to receive DNA fragments terminated at different ones of the four nucleic acid molecules, A, G, C and T respectively; characterized by advancing aparatus (47A, 53) for moving fragments from the end (31A) of said separating apparatus prior to the resolution of the last to be introduced bands while maintaining four separate groups of DNA fragments in accordance with the terminating base.
Claim 3
Dependent Claim
118 words
Apparatus according to claim 1 further characterized by means for marking said' DNA fragments prior to their entrance into the electrophoresis apparatus with biotin (11); means for attaching a fluorescent marker to avidin; (30A, 30G, 30C, 30T); means for marking said DNA fragments with fluorescenated avidin (FIG. 4); means for selectively pulsing said markers with light (52, FIG.6); means (64) for detecting the fluorescence of said marker across a time period in which background fluorescence is reduced; and said means for detecting including means for converting said fluorescence into electrical signals indicating the time sequence of bands of DNA marked with fluorescence corresponding to each of the DNA fragments cleaved at different ones of the nucleic acid groups.
Claim 4
Independent Claim
144 words
A method for sequencing DNA in which cloned DNA strands are prepared and cleaved into fragments having multiple bases per fragment; dividing the fragmented DNA strands into at least four batches with terminations at adenine bases and at random lengths respectively and applying samples from each of the four batches after terminating at their respective bases to four identical channels of separating apparatus (26A, 26G, 26C, 26T) characterized by separating the strands within the channels so that the first bands to be moved completely through the channels are fully resolved while the last bands are unresolved in a continuous process such that at least ten percent of the bands are resolved and moved through the channels while the least mobile bands are yet unresolved near the entrance end of the channel; and identifying and recording the time sequence bands in each of the channels.
Claim 5
Independent Claim
144 words
A method for sequencing DNA in which cloned DNA strands are prepared and cleaved into fragments having multiple bases per fragment; dividing the fragmented DNA strands into at least four batches with terminations at adenine bases and at random lengths respectively and applying samples from each of the four batches after terminating at their respective bases to four identical channels of separating apparatus (26A, 26G, 26C, 26T) characterized by separating the strands within the channels so that the first bands to be moved completely through the channels are fully resolved while the last bands are unresolved in a continuous process such that at least ten percent of the bands are resolved and moved through the channels while the least mobile bands are yet unresolved near the entrance end of the channel; and identifying and recording the time sequence bands in each of the channels.
Claim 6
Dependent Claim
68 words
A method according to claim 3 in which the step of separating includes the step of separating the strands by gel electrophoresis, HPLC or capillary electrophoresis with the terminating being by cleaving the strands at adenine, corresponding one of guanine, cytosine and thymine on pieces with random lengths for corresponding ones of the four channels characterized in that strands terminating at different bases are identified by terminating base.
Claim 7
Dependent Claim
66 words
A method according to claim 3 or claim 4 characterized in that the DNA strands are marked on one end with biotin before separating; and the fluorescent markers are attached to the avidin; by moving said bands sequentially from the gel while maintaining each fragment in each channel separate into means for marking with fluorescent-marked aviden by combining the fluorescent avidin with the biotin end markers.
Claim 8
Independent Claim
144 words
A method according to any of claims 3-5 characterized by moving the bands in sequence through a medium; scanning said bands with laser light having a narrow band width substantially conforming to the optimum absorption spectrum of the fluorescent markers; pulsing the laser light with pulses of shorter duration than three nanoseconds; detecting the fluorescent emission from the marker across a narrow selective band width conforming subtantially to the optimum emission spectrum of the markers through a second period of time; said second period of time beginning at least fifty nanoseconds from the start of its corresponding pulse of laser light and terminating at a time no greater than one hundred fifty nanoseconds from the start of the pulse of the laser light; and identifying and recording the time sequence of each of the channels so as to indicate the sequence of DNA fragments.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.