Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-12-04
2004-12-07
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200
Reexamination Certificate
active
06828100
ABSTRACT:
The present invention relates to methods of nucleic acid sequencing and in particular to sequencing-by-synthesis methods, ie. those methods based on the detection of nucleotide incorporation during polymerase extension, rather than on analysis of the nucleotide sequence itself, and to the improvements derivable in such methods by the use of a single-stranded DNA binding protein.
DNA sequencing is an essential tool in molecular genetic analysis. The ability to determine DNA nucleotide sequences has become increasingly important as efforts have commenced to determine the sequences of the large genomes of humans and other higher organisms. The two most commonly used methods for DNA sequencing are the enzymatic chain-termination method of Sanger and the chemical cleavage technique of Maxam and Gilbert. Both methods rely on gel electrophoresis to resolve, according to their size, DNA fragments produced from a larger DNA segment. Since the electrophoresis step as well as the subsequent detection of the separated DNA-fragments are cumbersome procedures, a great effort has been made to automate these steps. However, despite the fact that automated electrophoresis units are commercially available, electrophoresis is not well suited for large-scale genome projects or clinical sequencing where relatively cost-effective units with high throughput are needed. Thus, the need for non-electrophoretic methods for sequencing is great and several alternative strategies have been described, such as scanning tunnel electron microscopy (Driscoll et al., 1990, Nature, 346, 294-296), sequencing by hybridization (Bains et al., 1988, J. Theo. Biol. 135, 308-307) and single molecule detection (Jeff et al., 1989, Biomol. Struct. Dynamics, 7, 301-306), to overcome the disadvantages of electrophoresis.
Techniques enabling the rapid detection of a single DNA base change are also important tools for genetic analysis. In many cases detection of a single base or a few bases would be a great help in genetic analysis since several genetic diseases and certain cancers are related to minor mutations.
Sequencing-by-synthesis methods are useful ways of determining the sequence of a DNA molecule of up to a hundred or more bases or the identity of a single nucleotide within a sample DNA molecule. During typical sequencing-by-synthesis methods the four different nucleotides (adenine, thymine, guanine and cytosine) are conveniently added cyclically in a specific order; when the base which forms a pair (according to the normal rules of base pairing, A-T and C-G) with the next base in the single-strand target sequence is added, it will be incorporated into the growing complementary strand by a polymerase and this incorporation will trigger a detectable signal. The event of incorporation can be detected directly or indirectly. In direct detection, nucleotides are usually fluorescently labelled allowing analysis by a fluorometer. (U.S. Pat. No. 48,638,449, U.S. Pat. No. 5,302,509, Metzker et al. Nucl. Acids Res. (1994) 22: 4259-4267, Rosenthal International Patent Application No. WO 93/213401, WO 91/06678, Canard et al. Gene (1994) 148: 1-6). One such strategy of sequencing-by-synthesis called base addition sequencing scheme (BASS) is based on nucleotide analogues that terminate DNA synthesis. BASS involves repetitive cycles of incorporation of each successive nucleotide, in situ monitoring to identify the incorporated base, and deprotection to allow the next cycle of DNA synthesis.
Indirect detection usually takes advantage of enzymatic detection, e.g. measuring the release of PPi (inorganic pyrophosphate) during a polymerization reaction (WO 93/23564 and WO 89/09283). As each nucleotide is added to a growing nucleic acid strand during a polymerase reaction, a pyrophosphate molecule is released. It has been found that pyrophosphate released under these conditions can be detected enzymatically e.g. by the generation of light in the luciferase-luciferin reaction. Such methods enable a base to be identified in a target position and DNA to be sequenced simply and rapidly whilst avoiding the need for electrophoresis and the use of harmful radiolabels. These methods based on release of PPi are referred to herein as Pyrosequencing. The basic PPi-based sequencing methods have been improved by using a dATP analogue in place of dATP (WO 98/13523) and including a nucleotide-degrading enzyme such as apyrase during the polymerase reaction step, so that unincorporated nucleotides are degraded, as described in WO 98/28440.
However, these sequencing-by-synthesis methods mentioned above are not without drawbacks. A particular problem arises when the DNA to be sequenced has a number of identical adjacent bases, especially 3 or more the same.
FIG. 1
shows the trace obtained when a single-stranded PCR product is sequenced according to known sequencing-by-synthesis methods (in this case involving detection of PPi).
FIG. 1
shows that known methods do not provide clear results when two or more adjacent bases in the sample molecule are the same. For example, the peak height when the first set of three adenine residues are incorporated is almost the same as when four thymine residues are incorporated later; the set of three adenine residues incorporated around the middle of the sequence have the same peak height as previous doublets and the last pair of adenine residues to be incorporated have a peak height corresponding to single bases from the earlier part of the sequence.
Other problems of sequencing-by-synthesis methods include false signals which are the result of mispriming, i.e. hybridisation of the primer not to its targeted complement within the target DNA sequence but to another region which will result in generation of “incorporation signals” which do not reflect the identity of the target sequence. There is an associated problem which can result in a false indication of incorporation termed “minus frame incorporation”, where a proportion of the growing primer originating strands are not fully extended and false positive signals appear in subsequent cycles.
Thus, there is a need further to improve sequencing-by-synthesis methods by addressing the above problems and more generally to improve the accuracy of the methods while providing methods which are simple and quick to perform, lending themselves readily to automation.
It has surprisingly been found that including a single-stranded nucleic acid binding protein in the reaction mixture improves the ratio of signals generated by one, two, three or more adjacent bases and reduces the number of false signals and generally improves the efficacy and reduces the cost of sequencing-by-synthesis methods.
In one aspect, the present invention thus provides a method of identifying a base at a target position in a sample nucleic acid sequence wherein a primer, which hybridises to the sample nucleic acid immediately adjacent to the target position, is provided and the sample nucleic acid and primer are subjected to a polymerase reaction in the presence of a nucleotide whereby the nucleotide will only become incorporated if it is complementary to the base in the target position, and said incorporation is detected, characterized in that, a single-stranded nucleic acid binding protein is included in the polymerase reaction step.
The nucleic acid to be sequenced may be any nucleotide sequence it is desirable to obtain sequence information about. Thus, it may be any polynucleotide, or indeed oligonucleotide sequence. The nucleic acid may be DNA or RNA, and may be natural, isolated or synthetic. Thus, the target DNA may be genomic DNA, or cDNA, or a PCR product or other amplicon etc. Alternatively, the target DNA may be synthetic, and genomic DNA, cDNA or a PCR product etc. may be used as primer. The target (sample) nucleic acid may be used in any convenient form, according to techniques known in the art e.g. isolated, cloned, amplified etc., and may be prepared for the sequencing reaction, as desired, according to techniques known in the art.
The DNA may also be single or double-stranded whilst a si
Biotage AB
Dorsey & Whitney LLP
Horlick Kenneth R.
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