Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-09-05
2003-04-15
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C514S04400A, C536S023100, C536S024200, C536S024300, C536S024330, C536S024500
Reexamination Certificate
active
06548251
ABSTRACT:
BACKGROUND OF THE INVENTION
Oligonucleotides are widely used in DNA technologies. One of the most important properties of an oligonucleotide is its ability to bind to a complementary sequence in other polynucleotides. Robust and specific annealing of an oligonucleotide to its complementary sequence is important for the success of probe hybridization methods that allow detection and quantification of pathogens, genomic mutations and other nucleotide sequences. Unfortunately, some oligonucleotides composed of the naturally occurring nucleotides cannot be used as robust probes. For example, an oligonucleotide containing two segments of sequences that are complementary to each other (e.g., C
AAAAAAAAAA
CAC
TTTTTTTTTT
(SEQ ID NO: 67)) would form an internal structure called a hairpin that would prevent hybridization to its target. A further example is an oligonucleotide that can form a dimer with its second copy (e.g., ACTGAGACT
CTAATCGATTAG
(SEQ ID NO: 68)). Thus, there is a need for a method to inhibit the formation of such undesired structures.
Another typically unwanted biological or molecular process is the annealing of an oligonucleotide to non-target sequences in polynucleotides, called non-specific hybridization. This process increases the background signal in probe hybridization that limits the applications of this method and may lead to false positive results. The discrimination between specific and non-specific hybridization is most challenging when polynucleotides contain sequences that are similar to the target sequence. Another challenging situation is when very long polynucleotides (e.g., genomic DNA of 1 million (1 Mb) to 3 billion (3 Gb) base pairs) with a large amount of potential non-specific targets are present. Thus, there is a need for a method to inhibit non-specific hybridization of oligonucleotides.
There is a relatively narrow range of conditions (temperature, concentrations of ions and denaturing reagents) at which an oligonucleotide anneals specifically to its complementary target. These conditions are usually determined by measuring melting temperature (T
m
) of a duplex comprising an oligonucleotide and the second oligonucleotide that contains a sequence of bases complementary to the first oligonucleotide. Unfortunately, the range of conditions for the specific annealing of an oligonucleotide may not coincide with other requirements of the intended method. A common practice to meet these requirements is to select the length and GC content of an oligonucleotide probe with appropriate melting temperature. This selection may contradict other requirements on the length of an oligonucleotide. For example, a 40-mer oligonucleotide that has only one complementary sequence in a genomic DNA sequence generally has too high of a melting temperature and would anneal to partially complementary targets while a 15-mer oligonucleotide that has a suitable melting temperature would have too many complementary sequences in a genomic DNA sequence. Thus, there is a need for a method to inhibit non-specific hybridization of oligonucleotides at the wide variety of stringency conditions dictated by the requirements, other than the melting temperature, of nucleotide sequences.
Oligonucleotides and complexes with other polynucleotides are widely used as substrates for protein binding and enzymatic reactions. The enzymatic reaction typically results in chemical modification of an oligonucleotide, including cleavage of the oligonucleotide or addition of extra nucleotide(s). The latter reaction may be catalyzed by polymerase that uses an oligonucleotide as a primer and adds bases complementary to the bases in the template polynucleotide. Polymerase may also use an oligonucleotide as a template for polymerization reaction. Enzymatic reactions involving oligonucleotides constitute the core of many DNA technologies, for example, PCR, DNA sequencing, and SNP detection. The formation of undesired structures by an oligonucleotide or its complexes with other polynucleotides may interfere with the intended enzymatic reaction. Moreover, even transient formation of such undesired structures in a minute fraction of oligonucleotides could be amplified by the enzymatic reaction. One example of such an undesired process is the non-specific amplification by PCR that is difficult to avoid if the number of amplification cycles exceeds 40. Another such example is primer-dimer amplification during PCR. Thus, there is a need for a method to inhibit the ability of oligonucleotides to form such undesired structures in enzymatic reactions.
Oligonucleotides may serve different functions in DNA technologies that involve enzymatic reactions. One example is as a probe for detection of specific sequences amplified by PCR with two primers in a TaqMan assay. Such a probe should specifically bind to its complementary sequence and potential polymerization of the probe should be inhibited. Thus, under such circumstances, there is a need for a method to inhibit non-specific hybridization of an oligonucleotide and to inhibit its ability to function as a primer.
Oligonucleotides are also used as primers in primer extension reactions for SNP detection, which comprises one or more cycles of adding, by action of DNA polymerase, a labeled nucleotide to a primer annealed to its target complementary sequence. The results of this method would be jeopardized if the primer extension occurs at sites of non-specific annealing of the primer or if the primer itself serves as a template. For example, a hairpin C
AAAAAAAAAA
CAC
TTTTTTTTTT
(SEQ ID NO: 67) and dimer of ACTGAGACT
CTAATCGATTAG
(SEQ ID NO: 68) oligonucleotides could serve as templates and the resulting undesired products will be C
AAAAAAAAAA
CAC
TTTTTTTTTT
g (SEQ ID NO: 69) and ACTGAGACT
CTAATCGATTAG
a (SEQ ID NO: 70).
Oligonucleotides are also used as primers in primer extension reactions for DNA sequencing, which comprises one or more cycles of adding nucleotides, by action of DNA polymerase, to a primer annealed to its target complementary sequence and terminating the extension reaction at a specific base encoded in the template. The undesired processes described in the previous paragraph would jeopardize the results of this method. Undesired primer extension products may have additional bases at their 3′ ends and potentially could prime the reaction from targets that are complementary to the newly formed primers rather than to the original primers. In addition, polynucleotide products generated by the original primer extension could serve as templates for the annealing of the second copy of the primer and its subsequent extension. Should this event occur, it could generate a polynucleotide that has a primer sequence at its 5′ end and a sequence complementary to the primer at its 3′ end. DNA polymerase would generate the latter sequence at the final steps of the extension of the second copy of the primer when the nucleotides that comprise the first copy of the primer serve as templates. This polynucleotide would trigger exponential amplification (non-specific PCR) in a cycle sequencing method based on linear multiplication of products. Eventually, non-specific exponential amplification would overwhelm the linear multiplication and jeopardize the outcome of DNA sequencing. This undesired process limits the utility of such a cycle sequencing method. Thus, there is a need for a method to inhibit non-specific hybridization of an oligonucleotide, while retaining its ability to function as a primer and inhibiting its ability to function as a template in a polymerization reaction.
Oligonucleotides are also used as primers in primer extension reactions for PCR amplification, which comprises several cycles of adding nucleotides, by action of DNA polymerase, to primers annealed to target complementary sequences and termination of the extension reaction at the template end that is composed of nucleotides of another primer. The final 3′ end nucleotide added by DNA polymerase is complementary to the 5′ nucleotide of the other primer, and the fi
Kozyavkin Sergei A.
Malykh Andrei G.
Polouchine Nikolai N.
Slesarev Alexei I.
Fidelity Systems, Inc.
Myers Carla J.
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