Nucleotide compositions comprising photocleavable markers and methods of preparation thereof

#The present invention generally relates to nucleotides and polynucleotides useful in the sequencing of nucleic acids. The present invention specifically relates to compositions comprising nucleotides and polynucleotides comprising photocleavable labels and the methods of preparing said compositions.

#The sequencing of nucleic acids is one the most powerful and valuable tools for scientific research. As evidenced by the Human Genome project, there is an ever increasing demand for nucleic acid sequence information. There are numerous methods available for sequencing of nucleic acids. The first methods were developed almost twenty years ago. For example, the Sanger enzymatic (i.e., dideoxy chain termination) method involves synthesis of a DNA strand from a single-stranded template by a DNA polymerase. The Maxam and Gilbert method involves chemical degradation (i.e. chemical cleavage) of the original DNA. Both methods produce populations of radio-labelled polynucleotides that begin at a fixed point in the DNA to be sequenced and terminate at points which are dependent upon the location of a particular base in the original DNA strand. These polynucleotides are separated by a polyacrylamide gel electrophoresis, and the order of the nucleotides in the original DNA is directly read from an autoradiograph of the gel. However, the time-consuming electrophoresis step associated with these methods is difficult to perform in a highly parallel (i.e. greater than 1000 samples at a time per instrument) fashion.

#Although both the Sanger and Maxam-Gilbert methods are currently used, there have been many changes and improvements. The enzymatic chain termination method is probably the most popular and widely used technique for sequence determination, especially since the automation of the procedure has been accomplished through use of fluorescent, rather than radioactive labelling, and the utilization of amplification technology. The incorporation of amplification technology (e.g., the polymerase chain reaction [PCR]) enables the sequencing reaction to be cycled. Other advances include sequencing by chemiluminescence, multiplexing, and solid phase sequencing.

#FIG. 1 depicts one example of the general structure of the photocleavable marker-nucleotide conjugates of the present invention, compounds of general formula (I), wherein X is selected from the group consisting of a phosphate group and a hydrogen atom, M is a photocleavable marker, B is a nucleobase, and S is a sugar moiety.

#FIG. 2 shows one example of the incorporation of a photocleavable marker-nucleotide conjugate into polynucleic acid, detection of the marker, and removal of the marker by photocleavage, subsequent incorporation of the same (or a different) photocleavable marker-nucleotide conjugate into the same polynucleic acid and its subsequent detection, followed by separation on denaturing polyacrylamide gel and fluorescence imaging.

#FIG. 3 depicts one example of a synthesis scheme for BODIPY-FL-PC-aadUTP (compound 6) and Cy5-PC-aadUTP (compound 7).

#Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

#As used herein, the term “marker” refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a nucleotides, polynucleotides, or nucleic acids (including polynucleic acids). Markers may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Such markers can be added to the nucleotides and polynucleotides of the present invention. Marker molecules are “capable of being independently detected” where, in a mixture comprising two or more different markers, each marker has a separate and distinct detectable (preferably quantifiable) signal. For example, the present invention contemplates a photocleavable marker-polynucleotide conjugates comprising a plurality of different photocleavable marker molecules wherein each molecule emits a distinct signal only at a specific wavelength of UV light.

#Various methods of adding markers to nucleotides, polynucleotides, or nucleic acids are known in the art and may be used. Examples of markers for nucleotides, polynucleotides, or nucleic acids include, but are not limited to, the following: radioisotopes (e.g. 3H), fluorescent markers (e.g. BODIPY, Cy5, Cy3, FITC, rhodamine, and lanthanide phosphors), enzymatic markers (e.g. horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), biotinyl groups, pre-determined polypeptide epitopes recognized by a secondary reporter (e.g. leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, and epitope tags). In some embodiments, markers are attached by linkers, or spacer arms, of various lengths to reduce potential steric hindrance.