Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-09-16
2001-07-03
Kunz, Gary L. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025320, C536S025340, C536S026100, C536S026200, C536S026260
Reexamination Certificate
active
06255475
ABSTRACT:
The present invention relates to novel nucleic acid chain extension terminators, their use in nucleic acid sequencing and synthesis, respectively, as well as a method for preparing such compounds.
Today, there are two predominant methods for DNA sequence determination: the chemical degradation method (Maxam and Gilbert, Proc. Natl. Acad. Sci. 74:560-564 (1977), and the dideoxy chain termination method (Sanger et al., Proc. Natl. Acad. Sci. 74:5463-5467 (1977)). Most automated sequencers are based on the chain termination method utilizing fluorescent detection of product formation. In these systems either primers to which deoxynucleotides and dideoxynucleotides are added are dye-labelled, or the added dideoxynucleotides are fluorescently labelled. As an alternative, dye labelled deoxynucleotides can be used in conjunction with unlabeled dideoxynucleotides. This chain termination method is based upon the ability of an enzyme to add specific nucleotides onto the 3′ hydroxyl end of a primer annealed to a template. The base pairing property of nucleic acids determines the specificity of nucleotide addition. The extension products are then separated electrophoretically on a polyacrylamide gel and detected by an optical system utilizing laser excitation.
Although both the chemical degradation method and the dideoxy chain termination method are in widespread use, there are many associated disadvantages. For example, the methods require gel-electrophoretic separation. Typically, only 400-800 base pairs can be sequenced from a single clone. As a result, the systems are both time- and labor-intensive. Methods avoiding gel separation have been developed in attempts to increase the sequencing throughput.
Sequencing by hybridization (SBH) methods have been proposed by Crkvenjakov (Drmanac et al., Genomics 4:114 (1989); Strezoska et al., (Proc. Natl. Acad. Sci. USA 88:10089 (1991)), Bains and Smith (Bains and Smith, J. Theoretical Biol. 135:303 (1988)) and in U.S. Pat. No. 5,202,231. This type of system utilizes the information obtained from multiple hybridizations of the polynucleotide of interest, using short oligonucleotides to determine the nucleic acid sequence. These methods potentially can increase the sequence throughput beacuse multiple hybridization reactions are performed simultaneously. To reconstruct the sequence, however, an extensive computer search algorithm is required to determine the most likely order of all fragments obtained from the multiple hybridizations.
The SBH methods are problematic in several respects. For example, the hybridization is dependent upon the sequence composition of the duplex of the oligonucleotide and the polynucleotide of interest, so that GC-rich regions are more stable than AT-rich regions. As a result, false positives and false negatives during hybridization detection are frequently present and complicate sequence determination. Furthermore, the sequence of the polynucleotide is not determined directly, but is inferred from the sequence of the known probe, which increases the possibility for error.
Methods have also been proposed which detect the addition or removal of single molecules from a DNA strand.
For example, Hyman E. D., Anal. Biochem., 174:423 (1988) discloses the addition of a nucleotide to a an immobilised DNA template/primer complex in the presence of a polymerase and determination of polymerisation reaction by detecting the pyrophosphate liberated as a result of the polymerisation.
Jett et al., J. Biomol. Struct. Dyn., I, p. 301, 1989 discloses a method wherein a single stranded DNA or RNA molecule of labelled nucleotides, complementary to the sequence to be determined, is suspended in a moving flow stream. Individual bases are then cleaved sequentially from the end of the suspended sequence and determined by a detector passed by the flow stream.
EP-A-223 618 discloses the use of an immobilised DNA template, primer and polymerase exposed to a flow containing only one species of deoxynucleotide at a time. A downstream detection system then determines whether deoxynucleotide is incorporated into the copy or not by detecting the difference in deoxynucleotide concentrations entering and leaving the flow cell containing the complex of DNA template and polymerase.
WO 90/13666 proposes a method directly measuring the growth of the template copy rather than determining it indirectly from compositions in the flow medium. Only one of the four nucleotides is present at a time, and the polymerisation events reflecting the incorporation of a nucleotide or not are detected by spectroscopic means (evanescent wave spectroscopy, fluorescence detection, absorption spectroscopy) or by the individual nucleotides being labelled.
Similar methods employing labelled 3′-blocked deoxynucleotides where the blocking group is removable and which thus permit sequential deoxynucleotide addition/detection steps are disclosed in WO 91/06678, U.S. Pat. No. 5,302,509, DE-A-414 1178 and WO 93/21340. However, the necessary 3′-blocking groups are either not described in any detail, or are not accepted by the required enzyme, or do not permit desired rapid deblocking of the growing template copy strand after each polymerisation event.
One object of the present invention is to provide novel nucleotide derivatives which may be used as chain terminators and which by deprotection may readily be converted into nucleotides or nucleotide analogues that may be further extended.
Another object of the present invention is to provide a method for nucleotide sequence determination using the novel chain terminators.
Still another object of the present invention is to provide a method of synthesizing oligo- or polynucleotides by means of the novel chain terminators.
Another object of the present invention is to provide a process of preparing novel chain terminators according to the invention.
In accordance with the invention, these objects are achieved by the provision of a chain terminating nucleotide or nucleotide analogue having its 3′-hydroxyl group protected by an acetal or thioacetal structure designed in such a way that the 3′-hydroxyl can be deprotected in a relatively short time in dilute acid, such as hydrochloric acid at pH 2, for example.
In one aspect, the present invention therefore provides a compound of the general formula I:
or a salt thereof, such as a trimethylammonium, ammonium, sodium or potassium salt, wherein
B is a nucleobase,
X and Z independently are oxygen or sulphur,
Y is hydrogen, hydroxy or protected hydroxy, such as methoxy, ethoxy or allyloxy,
R
1
is hydrocarbyl, which optionally is substituted with a functional group,
R
2
is hydrogen or hydrocarbyl, which optionally is substituted with a functional group,
A is an electron withdrawing or electron donating group capable of moderating the acetal stability of the compound I via L
1
,
L
1
and L
2
are hydrocarbon linkers, which may be the same or different, L
2
, when present, being either (i) connected to L
1
via the group A, or (ii) directly connected to L
1
, the group A then being bound to one of linkers L
1
and L
2
,
F is a dye label,
Q is a coupling group for F, and
l, m and n independently are 0 or 1, with the proviso that l is 1 when m is 1, and l is 1 and m is 1 when n is 1.
In Formula I above, the nucleobase B may be natural or synthetic. Natural nucleobases include common nucleobases, such as adenine, guanine, cytosine, thymine and uracil, as well as less common nucleobases, such as xanthine, hypoxanthine or 2-aminopurine. Synthetic nucleobases B are analogues to the natural nucleobases and capable of interacting with other nucleobases in a specific, hydrogen bond determined way.
The hydrocarbyl groups represented by R
1
and R
2
include a wide variety, including straight and branched chain alkyl, alkenyl, aryl, aralkyl and cycloalkyl, preferably containing up to 10 carbon atoms. Preferred hydrocarbyl groups are primary, secondary or tertiary alkyl, alkenyl or alkynyl groups, especially lower alkyl groups, such as methyl and ethyl. The optional functional gr
Fish & Richardson P.C. P.A.
Kunz Gary L.
Owens Howard
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