3 blocked nucleic acid amplification primers

Details 3 blocked nucleic acid amplification primers 3 blocked nucleic acid amplification primers

2000-10-13

2003-01-21

Fredman, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S024300

Reexamination Certificate

active

06509157

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the field of molecular biology and nucleic acid chemistry. More specifically, it relates to methods and reagents for improving the yield of nucleic acid amplification reactions. The invention therefore has applications in any field in which nucleic acid amplification is used.
2. Description of Related Art
The invention of the polymerase chain reaction (PCR) made possible the in vitro amplification of nucleic acid sequences. PCR is described in U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188; Saiki et al., 1985, Science 230:1350-1354; Mullis et al., 1986, Cold Springs Harbor Symp. Quant. Biol. 51:263-273; and Mullis and Faloona, 1987, Methods Enzymol. 155:335-350; each of which is incorporated herein by reference. The development and application of PCR are described extensively in the literature. For example, a range of PCR—related topics are discussed in PCR Technology—principles and applications for DNA amplification, 1989, (ed. H. A. Erlich) Stockton Press, New York; PCR Protocols: A guide to methods and applications, 1990, (ed. M. A. Innis et al.) Academic Press, San Diego; and PCR Strategies, 1995, (ed. M. A. Innis et al.) Academic Press, San Diego; each of which is incorporated herein by reference. Commercial vendors, such as Perkin Elmer (Norwalk, Conn.), market PCR reagents and publish PCR protocols.
Since the original publication of nucleic acid amplification, various primer-based nucleic acid amplification methods have been described including, but not limited to, Ligase Chain Reaction (LCR, Wu and Wallace, 1989
, Genomics
4:560-569 and Barany, 1991
, Proc. Natl. Acad. Sci. USA
88:189-193); Polymerase Ligase Chain Reaction (Barany, 1991
, PCR Methods and Applic
. 1:5-16); Gap-LCR (PCT Patent Publication No. WO 90/01069); Repair Chain Reaction (European Patent Publication No. 439,182 A2); 3SR (Kwoh et al., 1989
, Proc. Natl. Acad. Sci. USA
86:1173-1177; Guatelli et al., 1990
, Proc. Natl. Acad. Sci. USA
87:1874-1878; PCT Patent Publication No. WO 92/08800); NASBA (U.S. Pat. No. 5,130,238); and strand displacement amplification (U.S. Pat. No. 5,455,166). All of the above references are incorporated herein by reference. A survey of amplification systems is provided in Abramson and Myers, 1993, Current Opinion in Biotechnology 4:41-47, incorporated herein by reference.
Specificity of primer-based amplification reactions largely depends on the specificity of primer hybridization. Under the elevated temperatures used in a typical amplification, the primers hybridize only to the intended target sequence. However, amplification reaction mixtures are typically assembled at room temperature, well below the temperature needed to insure primer hybridization specificity. Under such less stringent conditions, the primers may bind non-specifically to other only partially complementary nucleic acid sequences or to other primers and initiate the synthesis of undesired extension products, which can be amplified along with the target sequence. Amplification of non-specific primer extension products can compete with amplification of the desired target sequences and can significantly decrease the efficiency of the amplification of the desired sequence.
One frequently observed type of non-specific amplification product is a template independent artifact of amplification reactions referred to as “primer dimer”. Primer dimer is a double-stranded fragment whose length typically is close to the sum of the two primer lengths and appears of occur when one primer is extended over the other primer. The resulting concatenation forms an undesired template which, because of its short length, is amplified efficiently.
Non-specific amplification can be reduced by reducing the formation of primer extension products prior to the start of the reaction. In one method, referred to as a “hot-start” protocol, one or more critical reagents are withheld from the reaction mixture until the temperature is raised sufficiently to provide the necessary hybridization specificity. In this manner, the reaction mixture cannot support primer extension during the time that the reaction conditions do not insure specific primer hybridization.
Manual hot-start methods, in which the reaction tubes are opened after the initial high temperature incubation step and the missing reagents are added, are labor intensive and increase the risk of contamination of the reaction mixture. Alternatively, a heat sensitive material, such as wax, can be used to separate or sequester reaction components, as described in U.S. Pat. No. 5,411,876, incorporated herein by reference, and Chou et al., 1992, Nucl. Acids Res. 20(7):1717-1723, incorporated herein by reference. In these methods, a high temperature pre-reaction incubation melts the heat sensitive material, thereby allowing the reagents to mix.
Another method of reducing the formation of primer extension products prior to the start of the reaction relies on the heat-reversible inactivation of the DNA polymerase. U.S. Pat. Nos. 5,773,258 and 5,677,152, both incorporated herein by reference, describe DNA polymerases reversibly modified by the covalent attachment of a modifier group. Incubation of the inactivated DNA polymerase at high temperature results in cleavage of the modifier-enzyme bond, thereby releasing an active form of the enzyme.
Non-covalent reversible inhibition of a DNA polymerase by DNA polymerase-specific antibodies is described in U.S. Pat. Nos. 5,338,671, incorporated herein by reference.
Non-specific amplification also can be reduced by enzymatically degrading extension products formed prior to the start of the reaction using the methods described in U.S. Pat. No. 5,418,149, which is incorporated herein by reference. The degradation of newly-synthesized extension products is achieved by incorporating into the reaction mixture dUTP and UNG, and incubating the reaction mixture at 45-60° C. prior to carrying out the amplification reaction. Primer extension results in the formation of uracil-containing DNA, which is degraded by UNG under the pre-amplification conditions. A disadvantage of this method is that the degradation of extension product competes with the formation of extension product and the elimination of non-specific primer extension product is likely to be less complete. An advantage of this method is that uracil-containing DNA introduced into the reaction mixture as a contamination from a previous reaction is also degraded and, thus, the method also reduces the problem of contamination of a PCR by the amplified nucleic acid from previous reactions.
Conventional techniques of molecular biology and nucleic acid chemistry, which are within the skill of the art, are fully explained fully in the literature. See, for example, Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins. eds., 1984); and a series, Methods in Enzymology (Academic Press, Inc.), all of which are incorporated herein by reference. All patents, patent applications, and publications mentioned herein, both supra and infra, are incorporated herein by reference.
SUMMARY OF THE INVENTION
The present invention provides methods and reagents for the in vitro amplification of a nucleic acid sequence using a primer-based amplification reaction which provide a simple and economical solution to the problem of non-specific amplification. The methods use oligonucleotide primers reversibly blocked at the 3′-hydroxy terminus which can be unblocked by incubation in the amplification reaction mixture at an elevated temperature. Non-specific amplification is reduced because the reaction mixture does not support primer extension until the temperature of the reaction mixture has been elevated to a temperature which insures primer hybridization specificity.
One aspect of the invention relates to kits for the in vitro amplification of a nucleic aci

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