In-line complete spectral fluorescent imaging of nucleic...

Details In-line complete spectral fluorescent imaging of nucleic... In-line complete spectral fluorescent imaging of nucleic...

2001-05-18

2002-12-17

Shaw, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

Apparatus

Including measuring or testing

C435S006120, C435S007100, C435S091200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330

Reexamination Certificate

active

06495363

ABSTRACT:

FIELD OF THE INVENTION
The present invention is in the field of nucleic acid sequencing, and particularly relates to the use of the fluorescent reagents in the sequencing of nucleic acid molecules. More specifically, the present invention is in the field of sequencing via fluorescent nucleic acid molecules immobilized to a microchip and apparatuses for the same.
BACKGROUND OF THE INVENTION
The most commonly used methods of nucleic acid sequencing are the dideoxy-mediated chain termination method, also known as the “Sanger Method” (Sanger et al.,
J. Molec. Biol.
94:441 (1975); see also Prober et al.,
Science
238:336-340 (1987), both herein incorporated by reference in their entirety) and the “chemical degradation method,” also known as the “Maxam-Gilbert method” (Maxam et al.,
Proc. Natl. Acad. Sci.
(
U.S.A.
) 74:560 (1977), herein incorporated by reference in its entirety). Such methods are disclosed in Maniatis et al.,
Molecular Cloning, a Laboratory Manual,
2
nd Edition,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); Zyskind et al.,
Recombinant DNA Laboratory Manual,
Academic Press, Inc., New York (1988), both herein incorporated by reference in their entirety.
Both the dideoxy-mediated method and the Maxam-Gilbert method of DNA sequencing require the prior isolation of the DNA molecule that is to be sequenced. The sequence information is obtained by subjecting the reaction products to electrophoretic analysis (typically using polyacrylamide gels). Thus, a sample is applied to a lane of a gel, and the various species of nested fragments are separated from one another by their migration velocity through the gel.
In response to the difficulties encountered in employing gel electrophoresis to analyze sequences, several alternative methods have been developed. In one such method, a solid phase array of nucleic acid molecules is employed. The array consists of combinatorial (i.e., random or pseudo-random) nucleic acid molecules. Chetverin et al. provides a general review of solid-phase oligonucleotide synthesis and hybridization techniques (Chetverin et al.,
Bio/Technology
12:1093-1099 (1994), herein incorporated by reference in its entirety).
Macevicz, for example, describes a method for determining nucleic acid sequence via hybridization with multiple mixtures of oligonucleotide probes. In accordance with this method, the sequence of a target polynucleotide is determined by permitting the target to sequentially hybridize with sets of probes having an invariant nucleotide at one position, and a variant nucleotides at other positions (U.S. Pat. No. 5,002,867, herein incorporated by reference in its entirety). The Macevicz method determines the nucleotide sequence of the target by hybridizing the target with a set of probes, and then determining the number of sites that at least one member of the set is capable of hybridizing to the target (i.e., the number of “matches”). This procedure is repeated until each member of sets of probes has been tested.
Beattie et al. have described a protocol for the preparation of terminal amine-derivatized 9-mer oligonucleotide arrays on ordinary microscope slides (Beattie et al.,
Molec. Biotech.
4:213-225 (1995), herein incorporated by reference in its entirety). These oligonucleotide arrays can hybridize DNA target strands of up to several hundred bases in length and can discriminate against mismatches.
Drmanac has described a method for sequencing nucleic acid by hybridization using nucleic acid segments on different sectors of a substrate and probes which discriminate between a one base mismatch (Drmanac EP 797683, herein incorporated by reference in its entirety). Gruber describes a method for screening a sample for the presence of an unknown sequence using hybridization sequencing (Gruber, EP 787183, herein incorporated by reference in its entirety).
In contrast to the “Sanger Method” and the “Maxam-Gilbert method,” which identify the entire sequence of nucleotides of a target polynucleotide, “microsequencing” methods determine the identity of only a single nucleotide at a “predetermined” site. Such methods have particular utility in determining the presence and identity of polymorphisms in a target polynucleotide.
Because single nucleotide polymorphisms constitute sites of variation flanked by regions of invariant sequence, their analysis requires no more than the determination of the identity of the single nucleotide present at the site of variation; it is unnecessary to determine a complete gene sequence for each patient. Several methods have been developed to facilitate the analysis of such single nucleotide polymorphisms.
The GBA™ Genetic Bit Analysis method disclosed by Goelet et al. (WO 92/15712, herein incorporated by reference in its entirety) is a particularly useful microsequencing method. In GBA™, the nucleotide sequence information surrounding a predetermined site of interrogation is used to design an oligonucleotide primer that is complementary to the region immediately adjacent to, but not including, the predetermined site. The target DNA template is selected from the biological sample and hybridized to the interrogating primer. This primer is extended by a single labeled dideoxynucleotide using DNA polymerase in the presence of at least two, and most preferably all four chain terminating nucleoside triphosphate precursors.
Mundy (U.S. Pat. No. 4,656,127, herein incorporated by reference in its entirety) discusses alternative microsequencing methods for determining the identity of the nucleotide present at a particular polymorphic site. Mundy's method employs a specialized exonuclease-resistant nucleotide derivative. A primer complementary to the allelic sequence immediately 3′-to the polymorphic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular exonucleotide-resistant nucleotide derivative present, then that derivative will be incorporated by a polymerase onto the end of the hybridized primer. Such incorporation renders the primer resistant to exonuclease, and thereby permits its detection. Since the identity of the exonucleotide-resistant derivative of the sample is known, a finding that the primer has become resistant to exonucleases reveals that the nucleotide present in the polymorphic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. Mundy's method has the advantage that it does not require the determination of large amounts of extraneous sequence data. It has the disadvantages of destroying the amplified target sequences, and unmodified primer and of being extremely sensitive to the rate of polymerase incorporation of the specific exonuclease-resistant nucleotide being used.
Cohen et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087, both of which are herein incorporated by reference in their entirety) discuss a solution-based method for determining the identity of the nucleotide of a polymorphic site. As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employed that is complementary to allelic sequences immediately 3′-to a polymorphic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymorphic site will become incorporated onto the terminus of the primer.
In contrast to the method of Cohen et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087), the GBA™ method of Goelet et al. can be conducted as a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase. It is thus easier to perform, and more accurate than the method discussed by Cohen. The method of Cohen has the significant disadvantage of being a solution-based extension method that uses labeled dideoxynucleoside triphosphates. In the Cohen method, the target DNA template is usually prepared by a DNA amplification reaction, such

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