Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-06
2001-04-03
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S008000, C435S091200, C435S091210, C536S063000, C536S024330
Reexamination Certificate
active
06210891
ABSTRACT:
This invention relates to a method of sequencing DNA, based on the detection of base incorporation by the release of pyrophosphate (PPi). In particular, the invention relates to a “real-time” sequencing method.
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. A mini-sequencing protocol based on a solid phase principle was described (Hultman, et al., 1988, Nucl. Acid. Res., 17, 4937-4946; Syvanen et al., 1990, Genomics, 8, 684-692). The incorporation of a radiolabeled nucleotide was measured and used for analysis of the three-allelic polymorphism of the human apolipoprotein E gene. However, radioactive methods are not well suited for routine clinical applications and hence the development of a simple non-radioactive method for rapid DNA sequence analysis has also been of interest.
Methods of sequencing based on the concept of detecting inorganic pyrophosphate (PPi) which is released during a polymerase reaction have been described (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 enzymically 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.
However, the PPi-based sequencing methods mentioned above are not without drawbacks. Firstly, it has been found that dATP used in the sequencing reaction (chain extension) interferes in the subsequent luciferase-based detection reaction, by acting as a substrate for the luciferase enzyme. In many circumstances, this interference severely limits the utility of the method.
Secondly whilst the PPi-based methods described above do represent an improvement in ease and speed of operation, there is still a need for improved methods of sequencing which allow rapid detection and provision of sequence information. In particular there is a need for “real-time” methods of sequencing which enable the sequence information to be revealed simultaneously with, or very shortly after the sequencing, chain extension, reaction.
We now propose a novel modified PPi-based sequencing method in which these problems are addressed and which permits the sequencing reactions to be continuously monitored in real-time, with a signal being generated and detected, as each nucleotide is incorporated. This is achieved by using an dATP analogue, in place of dATP, which does not interfere with the luciferase reaction, and by performing the chain extension and detection, or signal-generation, reactions substantially simultaneously by including the “detection enzymes” in the chain extension reaction mixture. This represents a departure from the approach reported in the PPi-based sequencing proposed above, in which the chain extension reaction is first performed separately as a first reaction step, followed by a separate “detection” reaction, in which the products of the extension reaction are subsequently subjected to the luciferin-luciferase based signal generation (“detection”) reactions.
In one aspect, the present invention thus provides a method of identifying a base at a target position in a single-stranded sample DNA sequence wherein an extension primer, which hybridises to the sample DNA immediately adjacent to the target position is provided and the sample DNA and extension primer are subjected to a polymerase reaction in the presence of a deoxynucleotide or dideoxynucleotide whereby the deoxynucleotide or dideoxynucleotide will only become incorporated and release pyrophosphate (PPi) if it is complementary to the base in the target position, any release of PPi being detected enzymically, different deoxynucleotides or dideoxynucleotides being added either to separate aliquots of sample-primer mixture or successively to the same sample-primer mixture and subjected to the polymerase reaction to indicate which deoxynucleotide or dideoxynucleotide is incorporated, characterised in that, the PPi-detection enzyme(s) are included in the polymerase reaction step and in that in place of deoxy- or dideoxy adenosine triphosphate (ATP) a dATP or ddATP analogue is used which is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for a said PPi-detection enzyme.
The term dideoxynucleotide as used herein includes all 2′-deoxynucleotides in which the 3′-hydroxyl group is absent or modified and thus, while able to be added to the primer in the presence of the polymerase, is unable to enter into a subsequent polymerisation reaction.
PPi can be determined by many different methods and a number of enzymatic methods have been described in the literature (Reeves et al., (1969), Anal. Biochem., 28, 282-287; Guillory et al., (1971), Anal. Biochem., 39, 170-180; Johnson et al., (1968), Anal. Biochem., 15, 273; Cook et al., (1978), Anal. Biochem. 91, 557-565; and Drake et al., (1979), Anal. Biochem. 94, 117-120).
It is preferred to use luciferase and luciferin in combination to identify the release of pyrophosphate since the amount of light generated is substantially proportional to the amount of pyrophosphate released which, in turn, is directly proportional to the amount of base incorporated. The amount of light can readily be estimated by a suitable light sensitive device such as a luminometer.
Luciferin-luciferase reactions to detect the release of PPi are well known in the art. In particular, a method for continuous monitoring of PPi release based on the enzymes ATP sulphurylase and luciferase has been developed by Nyren and Lundin (Anal. Biochem., 151, 504-509, 1985) and termed ELIDA (Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay). The use of the ELIDA method to detect PPi is preferred according to the present invention. The method may however be modified, for example by the use of a more thermostable luciferase (Kaliyama et al., 1994, Biosci. Biotech. Biochem., 58, 1170-1171). This method is based on the following reactions:
ATP sulphurylase
PPi+APS→ATP+SO
4
2−
luciferase
ATP+luciferin+O
2
→AMP+PPi+oxyluciferin+CO
2
+hv
(APS=adenosine 5′-phosphosulphate)
The preferred detection enzymes involved in the PPi detection reaction a
Nyren Pal
Ronaghi Mostafa
Uhlen Mathias
Baker & Botts
Chakrabarti Arun
Fredman Jeffrey
Pyrosequencing AB
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