Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-04-24
2001-06-19
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091100, C435S091200, C435S006120, C435S007100, C435S015000, C536S024200, C536S063000
Reexamination Certificate
active
06248567
ABSTRACT:
FIELD OF THE INVENTION
The invention is generally directed to molecular biology techniques. Specifically, the invention is related to a method for selectively amplifying nucleic acids.
DESCRIPTION OF THE RELATED ART
The polymerase chain reaction (PCR) is a powerful tool with which nucleic acids (typically DNA) can be exponentially amplified. The basic PCR techniques are described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis, et al. PCR is run in cycles, typically with three different temperatures: denaturation, annealing, and extension. Two-temperature PCR is also possible. At the denaturation temperature, DNA templates are denatured. At the annealing temperature, primers anneal to the DNA template. PCR uses a thermally stable polymerase and at least one set of primers (i.e., two primers), which are short pieces of DNA typically around 20 base pairs, and which are present in a quantity in excess of the template. At the extension temperature, the polymerase extends the primers to form new DNA molecules. Primers and the extension products (i.e., the PCR products) are then melted from the target DNA, and a new cycle of PCR can commence with both the original target and the newly synthesized PCR products serving as templates in the next cycle of PCR. Hence, the exponential amplification.
In general, primers are designed based on known template sequences. One primer primes the sense strand, and the other primes the complementary strand of the target DNA. PCR can be performed on a uniform target DNA (i.e., targets with the same sequence) or on mixed target DNAs, (i.e., targets with different intervening sequences flanked by conserved sequences). For mixed target DNAs, even mismatched primers function in the PCR if the sequence of the targets have enough complementarity to the mismatched primers.
PCR is such a powerful tool that even a single molecule of template can be amplified. Li, H. et al. (1988) Nature 335:414-417. Although it has been possible to amplify large amounts of a rare template, it has been more difficult to amplify rare templates when they are mixed with similar templates, especially when rare templates are mixed with similar templates that are much more abundant.
Thus a need still exists for a method that amplifies desired templates while excluding or preventing amplification of similar undesired templates. One further challenge to amplifying only desired DNA templates in a mixed population is that in some mixed populations, one population member is often over-represented. Thus, amplification of the templates of interest results in an over-abundance of the one population member and a dearth of amplification of other species, including novel species.
When first practiced, PCR was not a quantitative method. That is, the number of PCR products generated was not representative of the initial starting number of template DNA molecules. This is due to several factors including (1) amplification is exponential, (2) a plateau effect, (3) substrate depletion, and (4) a preference to amplify small DNA targets over large DNA targets. Several modifications have been made to the PCR to make the technique quantitative. See, for example, U.S. Pat. No. 5,213,961 to Bunn, et al., which describes a process for quantitating nucleic acid species in a sample; and U.S. Pat. No. 5,219,727 to Wang, et al., which describes a method for determining the amount of a template nucleic segment in a sample by polymerase chain reaction.
Quantitative PCR is a powerful tool when the goal is to quantitate template DNAs. However, quantitative PCR is not useful if the goal is to amplify only certain desired DNA templates in a mixed population of templates. Sequence-specific PCR (or allele-specific PCR), can be used when the majority (or all) of the potential DNAs are known. Sequence-specific primers can be designed to bind to and amplify only certain target DNAs. However, when the majority (or all) of the potential DNAs are not known, this technique cannot be used.
It is useful then to devise a way to inhibit amplification of certain population members, especially without sorting a sample before amplification. It is desirable to amplify some nucleic acids while, at the same time, inhibiting the amplification of other nucleic acids. Some methods have been developed to address this goal. For example, U.S. Pat. No. 5,759,822 to Chenchik, et al. describes a method for adapter-mediated suppression of PCR amplification, in which adapters are ligated to DNA fragments. The adapters on either end (i.e., terminus) of the fragment can anneal to each other to form suppressive “pan-like” double-stranded structures that suppress amplification of the fragments during PCR. This method, however, is useful only for small fragments of DNA with known terminal sequences, such as cDNA/mRNA hybrid molecules and restriction fragments.
However, none of the above-noted approaches yields a method that can exclude at least some of undesired DNA templates from amplification and, at the same time, amplify desired DNA templates which are related to the undesired DNA templates. Such a method, described herein, expands the applicability of PCR methods to include mixed DNA templates having desired and undesired DNA templates, even when the desired DNA templates are unknown.
SUMMARY OF THE INVENTION
Described herein is a method for selectively amplifying a desired DNA template in samples containing a mixture of undesired and desired DNA templates. The method amplifies desired DNA templates and inhibits the amplification of the undesired DNA templates. In particular, the method is useful when some of the desired DNA templates have unknown intervening sequences and when the sequences of the desired and undesired DNA templates are similar. In the development of this technology, two primer sets were used: a first primer set that is a universal primer set and a second primer set that is a competitor primer set. Each primer set has a forward and a reverse primer. The forward primers of both the universal and competitor primer sets bind to the same strand. The competitor primer set binds to the undesired DNA template and inhibits the amplification of the undesired DNA template and generates a smaller PCR product from the undesired DNA template, allowing a larger PCR product to be generated from the desired DNA template. This method for competitively inhibiting amplification of an undesired DNA template has been described in the art. However, the problem with the use of competitor primers to generate smaller, undesired DNA template-specific PCR products is that the energy in the PCR is directed towards amplifying the smaller, competitive PCR products (i.e., the undesired fragments) instead of the desired DNA template.
In the preferred embodiment, three primer sets are used: a universal primer set, a competitor primer set, and a terminator primer set. This embodiment results in the inhibition of undesired DNA template amplification without significant generation of competitive PCR products, allowing PCR amplification from the desired DNA template. Preferably, the terminator primers have modifications at the 5′ and 3′ ends of the DNA sequence. Preferably, the 5′ modification increases the primer-DNA template binding affinity and prevents the terminator primer from being displaced after it hybridizes to a DNA template. Preferably, the 3′ modification inhibits primer extension, thereby preventing the generation of a PCR product from the terminator primer. The 5′ and 3′ modifications result in terminator primers with exceptionally high affinity for the undesired DNA template. The terminator primers bind to the undesired DNA template at much higher temperatures than the universal primers and directly interfere with the PCR amplification of the undesired DNA template by the universal primers. The sequence of each terminator primer is both identical to a region of the undesired DNA template that is not in common with the desired DNA templates and is complementary to the sequence of the opposite competitor prime
Goodman Robert M.
Liles Mark R.
DeWitt Ross & Stevens S.C.
Jones W. Gary
Leone, Esq. Joseph T.
Taylor Janell E.
Wisconsin Alumni Research Foundation
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