Modified nucleotides and methods useful for nucleic acid...

Details Modified nucleotides and methods useful for nucleic acid... Modified nucleotides and methods useful for nucleic acid...

2001-05-08

2003-06-17

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S091200

Reexamination Certificate

active

06579704

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates generally to a method for obtaining nucleic acid sequence information. More specifically, the present invention provides certain modified nucleotides referred to as Simtides for use in nucleic acid sequencing reactions.
Nucleic acid sequencing is a critical analytical technique used in the field of molecular biology. The development of reliable methods for sequencing has led to great advances in the understanding of the organization of genetic information and has laid the foundation for the detailed analysis of the structure and function of genes. Several methods have been developed to determine the nucleotide sequence of nucleic acids.
Two general methods currently used to sequence DNA include the Maxam-Gilbert chemical degradation method (A. M. Maxham et al.,
Methods in Enzymology
65, 499-559 (1980)) and the Sanger dideoxy chain termination method (F. Sanger, et al.,
Proc. Natl. Acad. Sci. USA
74, 5463-5467 (1977)). Both of these techniques are detailed in
Molecular Cloning: A Laboratory Manual
(Sambrook, Fritsch, Maniatis, eds., Cold Spring Harbor Laboratory Press, 1989), the disclosure of which is incorporated herein by reference.
With the Maxam-Gilbert technique, DNA fragments are prepared through base-specific chemical cleavage of the piece of DNA to be sequenced. The piece of DNA to be sequenced is first 5′-end-labeled with
32
P and then divided into four portions. Each portion is subjected to a different set of chemical treatments designed to cleave DNA at positions adjacent to a given base (or bases). The result is that all labeled fragments will have the same 5′-terminus as the original piece of DNA and will have 3′-termini defined by the positions of cleavage. This treatment is performed under conditions that generate DNA fragments of convenient lengths for separation by gel electrophoresis.
With the Sanger technique, DNA fragments are produced through partial enzymatic copying (i.e., synthesis) of the piece of DNA to be sequenced. In the most common version, the piece of DNA to be sequenced is inserted, using standard techniques, into a “sequencing vector”, a large circular, single-stranded piece of DNA such as the bacteriophage M13. This becomes the template for the copying process. A short piece of DNA with a sequence complementary to a region of the template just upstream from the insert is annealed to the template to serve as a primer for the synthesis. In the presence of the four natural deoxyribonucleoside triphosphates (dNTP's), a DNA polymerase will extend the primer from the 3′-end to produce a complementary copy of the template in the region of the insert. To produce a complete set of sequencing fragments, four reactions are run in parallel, each containing the four dNTP's along with a single dideoxyribonucleoside triphosphate (ddNTP) terminator, one for each base.
32
P-labeled or fluorophore-labeled dNTP is added to afford labeled fragments. If a dNTP is incorporated by the polymerase, chain extension can continue. If the corresponding ddNTP is selected, the chain is terminated. The ratio of ddNTP to dNTP is adjusted to generate DNA fragments of appropriate lengths. Each of the four reaction mixtures will, thus, contain a distribution of fragments with the same dideoxynucleoside residue at the 3′-terminus and a primer-defined 5′terminus.
Fragments generated utilizing the Sanger method of sequencing may be end-labeled, via, for example, the utilization of primers having labeled nucleotides incorporated into their sequence. Alternatively, molecules may be end-labeled via the utilization of labeled dideoxynucleosides or other modified chain-terminating nucleotides or nucleotide mimics. Molecules can also be labeled internally by the utilization of one or more labeled nucleotides incorporated during the synthesis step of the process.
In both the Sanger and Maxam-Gilbert methods, base sequence information, which generally cannot be directly determined by physical methods, is converted into chain-length information, which can be determined. This determination can be accomplished through electrophoretic separation. Under denaturing conditions (e.g., high temperature, presence of urea, etc.), short DNA fragments migrate as if they were stiff rods. If a gel matrix is employed for the electrophoresis, the DNA fragments are sorted by size. The single-base resolution required for sequencing can usually be obtained for DNA fragments containing up to several hundred bases. To determine a full sequence, the four sets of fragments produced by either Maxam-Gilbert or Sanger methodology are subjected to electrophoresis. This results in the fragments being spatially resolved along the length of the gel.
Dyes such as, for example, infrared dyes, fluorescent dyes, colorimetric dyes, chemiluminescent dyes, and/or other detectable molecules, can be used instead of the
32
P label in the foregoing sequencing reactions. Molecules other than dideoxynucleotides may also be used as chain terminators in these reactions.
One method of discriminating dyes in these types of reactions is described in U.S. patent application Ser. No. 07/057,566 (Prober et al.) filed Jun. 12, 1987, abandoned, entitled “Method, System, and Reagents for DNA Sequencing”. This system is available from E.I. Du Pont de Nemours and Company (Wilmington, Del.), and is known as the Genesis™ 2000. The system comprises a means for detecting the presence of radiant energy from closely-related yet distinguishable reporters or labels that are covalently attached to compounds which function as chain-terminating nucleotides in a modified Sanger DNA chain-elongation method. Distinguishable fluorescent reporters are attached to each of the four dideoxynucleotide bases represented in Sanger DNA-sequencing reactions, i.e., dideoxynucleotides of adenine (A), guanine (G), cytosine (C), and thymine (T). These reporter-labeled chain-terminating reagents are substituted for unlabeled chain terminators in the traditional Sanger method and are combined in reactions with the corresponding deoxynucleotides, an appropriate primer, template, and polymerase. The resulting mixture contains DNA fragments of varying length that differ from each other by one base and terminate on the 3′-end with uniquely labeled chain terminators corresponding to one of the four DNA bases. This labeling method allows elimination of the customary radioactive label contained in one of the deoxynucleotides of the traditional Sanger method.
Detection of these reporter labels can be accomplished with two stationary photomultiplier tubes (PMT's) that receive differing wavelength bands of fluorescent emissions from laser-stimulated reporters attached to chain terminators on the DNA fragments. These fragments can be electrophoretically separated in space and/or time to move along an axis perpendicular to the sensing area of the PMT's. The fluorescent emissions first pass through a dichroic or other wavelength-selective filter or filters, placed so as to direct one characteristic wavelength to one PMT and the other characteristic wavelength to the other PMT. In this manner, different digital signals are created in each PMT that can be ratioed to produce a third signal that is unique to a given fluorescent reporter, even if a series of fluorescent reporters have closely-spaced emission wavelengths. This system is capable of detecting reporters with efficiently-spaced emissions whose maxima differ by only 5 to 7 nm. Therefore, the sequential base assignments in a DNA strand of interest can be made on the basis of the unique ratio derived for each of the four reporter-labeled chain terminators which correspond to each of the four bases in DNA.
Although the base information in the Genesis™ system is contained in fluorescent labels, the information may also be contained in colorimetric labels (S. Beck,
Anal. Biochem.
164(2), 514-520 (1987)), chemiluminescent labels (S. Beck
Nucleic Acids Research
17, 5115-5123 (1989)), or other labels.
The G

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