Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1995-07-21
2001-03-27
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091200, C536S022100, C536S025300, C536S025320
Reexamination Certificate
active
06207421
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to DNA terminators for signaling the end of DNA strand synthesis which may be used to prepare DNA strands and for sequencing and to the marking and sequencing of DNA strands.
In one class of techniques for sequencing DNA, identical strands of DNA are marked. The strands are separated into four aliquots. The strands in a given aliquot are either individually cleaved at or synthesized to any base belonging to only one of the four base types, which are adenine, guanine, cytosine and thymine (hereinafter A, G, C and T). The adenine-, guanine-, cytosine- and thymine-terminated strands are then electrophoresed for separation. The different marked strands are detected and the terminating base identified for each. The rate of electrophoresis of the different terminated base strands indicates the DNA sequence.
In one class of technique for marking DNA strands, the strands are marked with a fluorescent dye. The strands are marked by: (1) hybridzing specially synthesized fluorescently marked oligonucleotide primer strands to template strands of DNA and then extending the primer strands with DNA polymerase to incorporate unmarked deoxy nucleotide triphosphates and an unmarked terminator such as a dideoxynucleotide triphosphate for the purpose of uniquely terminating strand synthesis; (2) hybridizing unmarked oligonucleotide primer strands to template strands and then extending the primer strands with DNA polymerase to incorporate marked deoxynucleotide triphosphates and an unmarked terminator such as a dideoxy nucleotide triphosphate for the purpose of uniquely terminating strand syntesis; or (3) hybridizing unmarked oligonucleotide primer strands with DNA polymerase to incorporate unmarked deoxynucleotide triphosphates and a marked terminator such as a dideoxy nucleotide triphosphate for the purpose of uniquely terminating strand synthesis.
In a prior art technique of this class, the marked strands are of four types, each corresponding to the appropriate one of adenine-, guanine-, cytosine-, and thymine-terminated strands. Moreover, in prior art sequencing techniques of this class, the fluorescent dyes used as markers have their maximum emission spectra in the visible range with wavelengths shorter than 650 nm, the DNA is subject to irradiation in this visible spectra, and visible spectra detectors and light sources are used. Generally photomultipliers tubes are used for detection and gas lasers such as Argon-ion or Helium—Neon lasers are used as light sources.
Cyanine dyes are known to absorb far red (600-700 nm) and near infrared (700-1200 nm) light and techniques for the synthesis of derivatives of the cyanine dyes are known. The use of cyanine dyes as markers for DNA sequencing has been successfully achieved as described by Middendorf, L. R., et al., “Electrophoresis Journal” 13, 487-494 (1992).
An additional disadvantage of the prior art techniques for DNA sequencing is the need to have four different terminators, regardless of whether or not they are marked, for the purpose of providing a unique termination of strand synthesis.
In a prior art sequencing technique, the DNA strands are marked with a radioactive marker, and after being separated by electrophoresis, film is exposed to the gel and developed to indicate the sequence of the bands. The range of lengths and resolution of this type of static detection is limited by the size of the apparatus.
The prior art techniques for DNA sequencing have several disadvantages such as: (1) because the dyes have their emission spectra in that visible region of the light spectrum having a wavelength shorter than 650 nm, the lasers used to excite the fluorescent markers, and under some circumstances, the detectors for the light tend to be expensive; (2) the signal information is relatively noisy due to the background fluorescence interference by biomolecules and glass; (3) they are relatively slow; (4) they are at least partly manual; (5) they are limited to relatively short strands of DNA; (6) they do not have high sensitivity; and (7) they required complex protocols involving multiple types of synthesis terminators.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the invention to provide a novel technique for DNA termination.
It is a further object of the invention to provide a novel technique for marking DNA strands.
It is a still further object of the invention to provide a novel technique for DNA sequencing.
It is a still further object of the invention to provide a novel technique for terminating DNA strand synthesis.
It is a still further object of the invention to provide a novel universal terminator for DNA sequencing.
It is a still further object of the invention to provide novel apparatuses and methods for sequencing relatively large fragments of DNA.
It is a still further object of the invention to provide novel apparatuses and methods for sequencing DNA fragments of 100 bases or more.
It is a still further object of the invention to provide a technique for continuous sequencing of DNA.
It is a still further object of the invention to continuously sequence DNA without the spatial limitations of range of lengths and resolution.
It is a still further object of the invention to provide a novel technique for continuously sequencing DNA using fluorescent detection.
It is a still further object of the invention to provide a novel technique for continuously sequencing DNA marked with fluorescence which more clearly distinguishes marked DNA fragments from background fluorescence.
It is a still further object of the invention to provide a novel technique for scanning fluorescent material.
It is a further object of the invention to provide novel equipment and methods for the sequencing of far red and/or infrared but especially far red (600-700 nm) and/or near infrared (700-3000 nm) fluorescence labeled DNA and the detection of the DNA after irradiation by light from laser diodes.
It is a still further object of the invention to provide a novel technique for DNA sequencing using a novel fluorescent marker attached to the DNA.
It is a still further object of the invention to provide a novel fluorescent marker, method of synthesizing the marker and of attaching it to DNA.
It is a further object of the invention to provide a novel method, marker and equipment that permits the detection of marked molecules, including DNA, using diode light sources such as laser diodes or light emitting diodes and semiconductor detectors such as photodiodes or charge-coupled devices (CCD's).
In accordance with the above and further objects of the invention, strands of DNA are prepared and continuously electrophoresed and identified. For this purpose, the strands are fluorescently marked. The light emitted while irradiating the strands near the terminal end of the electrophoresis channel is detected and correlated. The electrophoresis conditions are selected so that strands being electrophoresed near the terminal end of the electrophoresis channel are fully resolved prior to the resolution of longer strands that have not yet reached the terminal end of the electrophoresis channel, and so on, in a continuous process over a period of time.
The gel size, electric field and DNA mobilities are such that the first bands to be moved completely through the gel are fully resolved while the last bands are yet unresolved in a continuous process controlled to cause at least ten percent of the bands to be resolved and electrophoresed through the gel while the less mobile bands are yet unresolved near the entrance end of the gel. These less mobile bands become resolved little by little over time in a continuous fashion without interruption of the movement of these bands through the gel.
In one embodiment for labeling strands, a substrate that terminates synthesis is labeled with a marker. In another embodiment, the strand is labeled either by a labeled primer that provides the initial portion of the strand or by a labeled deoxy nucleotide triphosphate that allows labeling of the strand during elongation. In any of
Brumbaugh John A.
Middendorf Lyle Richard
Carney Vincent L.
Li-Cor Inc.
Riley Jezia
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