De novo or “universal” sequencing array

Details De novo or “universal” sequencing array De novo or “universal” sequencing array

1997-11-21

2001-11-27

Houtteman, Scott W. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S024300, C435S287200

Reexamination Certificate

active

06322968

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a nucleic acid sequencing reagent based on a combinatorial array of 4-8 specific bases, combined with one or more random bases or other spacer molecules, and a “generic” template capture moiety capable of binding to some common region of the template nucleic acid. Template nucleic acids, such as DNA amplified by PCR, can be sequenced or scanned for mutations using this array configuration through primer extension with labeled ddNTPs. This array can also be used to sequence templates without prior knowledge (de novo) of the wild type or “expected” sequence.
BACKGROUND OF THE INVENTION
I. NUCLEIC ACID SEQUENCING
Initial attempts to determine the sequence of a DNA molecule were extensions of techniques which had been initially developed to permit the sequencing of RNA molecules (Sanger, F.,
J. Mol. Biol
. 13:373 (1965); Brownlee, G. G. et al.,
J. Mol. Biol
. 34:379 (1968)). Such methods involved the specific cleavage of DNA into smaller fragments by (1) enzymatic digestion (Robertson, H. D. et al.,
Nature New Biol
. 241:38 (1973); Ziff, E. B. et al.,
Nature New Biol
. 241:34 (1973)); (2) nearest neighbor analysis (Wu, R., et al.,
J. Mol. Biol
. 57:491 (1971)), and (3) the “Wandering Spot” method (Sanger, F.,
Proc. Natl. Acad. Sci
. (
U.S.A
.) 70:1209 (1973)).
The most commonly used methods of nucleic acid sequencing are the dideoxy-mediated chain termination method, also known as the “Sanger Method” (Sanger, F., et al.,
J. Molec. Biol
. 94:441 (1975); Prober, J. et al.,
Science
238:336-340 (1987)) and the “chemical degradation method,” also known as the “Maxam-Gilbert method” (Maxam, A. M., et al.,
Proc. Natl. Acad. Sci
. (
U.S.A
.) 74:560 (1977), both references herein incorporated by reference).
A. The Maxam-Gilbert Method Of DNA Sequencing
The Maxam-Gilbert method of DNA sequencing is a degradative method. In this procedure, a fragment of DNA is labeled at one end (or terminus) and partially cleaved in four separate chemical reactions, each of which is specific for cleaving the DNA molecule at a particular base (G or C) at a particular type of base (A/G, C/T, or A>C). As in the dideoxy method, the effect of such reactions is to create a set of nested molecules whose lengths are determined by the locations of a particular base along the length of the DNA molecule being sequenced. The nested reaction products are then resolved by electrophoresis, and the end-labeled molecules are detected, typically by autoradiography when a
32
P label is employed. Four single lanes are typically required in order to determine the sequence.
Significantly, in the Maxam-Gilbert method the sequence is obtained from the original DNA molecule, and not from an enzymatic copy. For this reason, the method can be used to sequence synthetic oligonucleotides, and to analyze DNA modifications such as methylation, etc. It can also be used to study both DNA secondary structure and protein-DNA interactions. Indeed, it has been readily employed in the identification of the binding sites of DNA binding proteins.
The Maxam-Gilbert method uses simple chemical reagents which are readily available. Nevertheless, the dideoxy-mediated method has several advantages over the Maxam-Gilbert method. The Maxam-Gilbert method is extremely laborious and requires meticulous experimental technique. In contrast, the Sanger method may be employed on larger nucleic acid molecules.
B. Dideoxy-Mediated Chain Termination Method Of DNA Sequencing
In the dideoxy-mediated or “Sanger” chain termination method of DNA sequencing, the sequence of a DNA molecule is obtained through the extension of an oligonucleotide primer which is hybridized to the nucleic acid molecule being sequenced. In brief, four separate primer extension reactions are conducted. In each reaction, a DNA polymerase is added along with the four nucleotide triphosphates needed to polymerize DNA. Each of these reactions is carried out in the additional presence of a 2′,3′ dideoxy derivative of the A, T, C, or G nucleoside triphosphate. Such derivatives differ from conventional nucleoside triphosphates in that they lack a hydroxyl residue at the 3′ position of deoxyribose. Thus, although they can be incorporated by a DNA polymerase into the newly synthesized primer extension, the absence of the 3′ hydroxyl group causes them to be incapable of forming a phosphodiester bond with a succeeding nucleoside triphosphate. The incorporation of a dideoxy derivative results in the termination of the extension reaction.
Because the dideoxy derivatives are present in lower concentrations than their corresponding, conventional nucleoside triphosphate analogs, the net result of each of the four reactions is to produce a set of nested oligonucleotides each of which is terminated by the particular dideoxy derivative used in the reaction. By subjecting the reaction products of each of the extension reactions to electrophoresis, it is possible to obtain a series of four “ladders.” Since the position of each “rung” of the ladder is determined by the size of the molecule, and since such size is determined by the incorporation of the dideoxy derivative, the appearance and location of a particular “rung” can be readily translated into the sequence of the extended primer. Thus, through an electrophoretic analysis, the sequence of the extended primer can be determined.
Methods for sequencing DNA using either the dideoxy-mediated method or the Maxam-Gilbert method are widely known to those of ordinary skill in the art. Such methods are, for example, disclosed in Maniatis, T. et al.,
Molecular Cloning, a Laboratory Manual
, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N. Y. (1989), and in Zyskind, J. W. et al.,
Recombinant DNA Laboratory Manual
, Academic Press, Inc., New York (1988), both of which are herein incorporated by reference.
Both the dideoxy-mediated method and the Maxam-Gilbert method of DNA sequencing require the prior isolation of the DNA molecule which 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.
C. Sequencing Via Hybridization To Ordered Oligonucleotide Arrays
In response to the difficulties encountered in employing gel electrophoresis to analyze sequences, alternative methods have been developed. Existing methods for de novo sequencing on solid phase arrays consist primarily of hybridization of template nucleic acids to arrayed primers containing combinatorial sequences which hybridize to complementary sequences on the template strand. These methods combine the capture of the template, by formation of stable duplex structures, with sequence discrimination due to instability of mismatches between the template and the primer. Chetverin, A. B. et al. provides a general review of solid-phase oligonucleotide synthesis and hybridization techniques. Chetverin, A. B. et al.,
Bio/Technology
12:1093-1099 (1994).
Macevicz (U.S. Pat. No. 5,002,867), 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. 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 a sets of probes has been tested.
Beattie, W. G. et al. has described a protocol for the preparation of terminal amine-derivatized 9-mer oligonucleotide arrays on ordinary

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