Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-09
2001-09-25
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100
Reexamination Certificate
active
06294336
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to nucleic acid chemistry, and more specifically to a method for determining the nucleotide sequence of a polynucleotide. The invention further relates to apparatus and kits that embody or employ such a method.
BACKGROUND OF THE INVENTION
The determination of the nucleotide sequence of a polynucleotide has substantial utility in medicine, forensics, biomedical research, and in the determination of paternity and identity. Several methods for determining the nucleotide sequence of a polynucleotide have been identified.
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 “Wanderings 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. 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 the reactions is carried out in the additional presence of a 2′,3′ dideoxy derivative of the A, T, C, or G nucleoside triphosphates. Such derivatives differ from conventional nucleotide 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 nucleotide triphosphate. Thus, the incorporation of a dideoxy derivative results in the termination of the extension reaction. Since the dideoxy derivatives are present in lower concentrations than their corresponding, conventional nucleotide 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.
One deficiency of the dideoxy-mediated sequencing method is the need to optimize the ratio of dideoxy nucleoside triphosphates to conventional nucleoside triphosphates in the chain-extension/chain-termination reactions. Such adjustments are needed in order to maximize the amount of information which can be obtained from each primer. Additionally, the efficiency of dideoxy nucleotide incorporation in a particular target molecule is partially dependent upon the primary and secondary structures of the target.
The dideoxy-mediated method thus requires single-stranded templates, specific oligonucleotide primers, and high quality preparations of a DNA polymerase (typically the Klenow fragment of
E. coli
DNA polymerase I). Initially, these requirements delayed the wide spread use of the method. However, with the ready availability of synthetic primers, and the availability of bacteriophage M13 and phagemid vectors (Maniatis, T., et al.,
Molecular Cloning, a Laboratory Manual.
2
nd Edition
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), herein incorporated by reference), the dideoxy-mediated chain termination method is now extensively employed.
B. 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 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 above-described 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.
The Maxam-Gilbert method thus 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.
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.
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.
2
nd 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 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. The number of nested fragments which can be separated in a single lane is approximately 200-300 regardless of whether the Sanger or the Maxam-Gilbert method is used. Those of great skill in the art can separate up to 600 fragments in a single lane. Thus, in order to sequence large DNA molecules, it is necessary to fragment the molecule, and to sequence the fragments in separate lanes of the sequencing gel. The sequence of the entire molecule is obtained by orienting and ordering the sequence data obtained from each fragment.
Two approaches have been employed by those of skill in this art to accomplish this goal. In a random or shotgun
Boyce-Jacino Michael T.
Goelet Philip
Rogers Yu-Hui
Horlick Kenneth R.
Kalow David A.
Kalow & Springut LLP
Orchid BioSciences, Inc.
Schmidt William D.
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