Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-28
2001-10-09
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06300075
ABSTRACT:
FIELD OF THE INVENTION
This invention pertains to methods for amplifying nucleic acids, particularly in diagnostic tests for infectious microorganisms.
BACKGROUND OF THE INVENTION
Amplification of nucleic acid sequences using polymerase chain reaction (PCR) requires at least two oligonucleotide amplification primers that hybridize to different sequences within the target nucleic acid. In general, it is desirable to avoid the use of oligonucleotide primers having homologous sequences at their 3′ ends. Primers with homologous 3′ ends can potentially hybridize to each other, resulting in a variety of amplification artifacts, including primer-dimers.
Hybridization of primers having 3′ end complementarity can occur once all of the PCR reaction components have been mixed, but prior to initiation of amplification, through stabilization of primer hybrids at low temperatures due to the presence of magnesium in the reaction mixture. Primer-dimer formation occurs at or below room temperature by extension of the hybridized primers by DNA polymerase. The resulting primer-dimer product will amplify during PCR, competing with target nucleic acid for primers and polymerase. If enough primer-dimer product is formed in the initial phase, subsequent PCR amplification of this product can out-compete the designated target, leading to either (i) a false negative result, i.e., the sample appears to lack a particular sequence when in fact the sequence is present or (ii) a “no-test” results, i.e., no signal is obtained from an internal positive control.
In addition to the primary sequence of the primer sets, the major contributors to primer-dimer formation are: a high molar level of primers in the assay specific master mix (ASMM); the order of addition of reactants (for example, adding the ASMM, then MgCl
2
solution, then target/sample will increase primer-dimer formation); the incubation period between addition of MgCl
2
to the ASMM and addition of target; and the time period during which a complete amplification reaction admixture, i.e., comprising all the components required for amplification, including target and polymerase, incubates prior to initiation of PCR thermocycling.
Several approaches are known to reduce primer-dimer formation in a nucleic acid amplification admixture of primers and polymerase. These approaches include:
1. Carefully designing the PCR primers to minimize 3′-end homologies with all other primers in a particular reaction mixture. However, this strategy is difficult in a multiplex reaction, i.e., a reaction containing several pairs of amplification primers directed to different target nucleic acid sequences. Furthermore, even with a single pair of primers, this may be difficult to achieve, due to other constraints, such as, e.g., regions of identity or conservation.
2. Performing the thermal cycling/amplification reaction soon after preparing an assay-specific reaction admixture to reduce the amount of time available for primer-dimer formation. However, this can be difficult in practice, particularly if large numbers of samples are to be screened.
3. Changing the order of reagent addition to destabilize potential primer-dimers. For example, adding MgCl
2
as the last component can reduce primer-dimer formation. This approach, however, is not desirable because (i) it does not eliminate primer-dimer formation, (ii) it is inconvenient, and (iii) it can result in contamination of the magnesium chloride solution with target from a sample.
4. Using “triggering” antibodies, which are anti-polymerase antibodies that block polymerase activity at temperatures where primer-hybrids may form, but which are inactivated at high temperatures. Thus, polymerase is only activated at temperatures that are too high for primer-dimers to form. (See, U.S. Pat. Nos. 5,338,671 and 5,587,287; and European Patent Application No. 0592035). However, since antibody/polymerase binding is an equilibrium process, complete binding of all polymerase cannot be achieved. Thus, antibody-based PCR triggering is not necessarily 100% effective, particularly in complex amplification reactions.
5. Performing Hot Start PCR (Chou et al.,
Nuc. Acids Res.
20:1717-1723, 1992). This involves adding everything except the thermostable DNA polymerase to the reaction admixture, initiating the reaction with a product denaturation step, followed by opening up the reaction tubes and adding the polymerase to the reaction admixture. Although this method is effective at reducing primer-dimer formation, it is not practical for a number of reasons. Most notably, it is very cumbersome and significantly increases the likelihood of amplicon carryover.
6. Performing Hot Start PCR using a thermostable DNA polymerase, such as AmpliTaq Gold, that is relatively inactive until heating (See, European Patent application No. 624641 and U.S. Pat. No. 5,491,086). However, AmpliTaq Gold retains significant residual enzyme activity at low temperatures, and thus is still prone to generating side products.
None of these approaches is predictably successful at eliminating primer-dimer formation. Careful primer design and the use of AmpliTaq Gold or triggering antibodies in combination with Taq Polymerase can reduce primer-dimer formation, but does not eliminate it. Thus, there is a need in the art for improved methods for reducing further or eliminating primer-dimer formation in PCR reactions, particularly in multiplex reactions.
SUMMARY OF THE INVENTION
The present invention provides methods for increasing the specificity of amplification of a target nucleic acid. It can be applied to all reactions requiring oligonucleotide amplification primers specific to the target and a nucleic acid polymerase. Thus, in one aspect the invention is directed to a method for increasing the specificity of amplification of a target nucleic acid in an amplification reaction, where the amplification reaction mixture comprises one or more oligonucleotide amplification primers specific to the target, a nucleic acid polymerase, and one or more magnesium salts, comprising preparing a primer-carrier admixture containing one or more primers and carrier nucleic acid, and contacting the primer-carrier admixture with the target nucleic acid, one or more magnesium salts, and polymerase.
In another aspect, the invention is directed to a method for increasing specificity of amplification of a target nucleic acid in an amplification reaction, where the amplification reaction mixture comprises one or more oligonucleotide amplification primers specific to the target nucleic acid, Taq polymerase, and magnesium chloride, comprising preparing a primer-carrier admixture including one or more primers and carrier nucleic acid including calf thymus DNA, where the concentration of the carrier nucleic acid will range from about 1 to about 100 micrograms/ml of amplification reaction mixture, and contacting, at a temperature less than about 100° C., the primer-carrier admixture with the target nucleic acid, Taq polymerase; and magnesium chloride.
In yet another aspect, the invention is directed to a method for reducing polymerize extension of non-target nucleic acids in a reaction for the amplification of a target nucleic acid, where the amplification reaction mixture comprises one or more oligonucleotide amplification primers specific to the target nucleic acid, polymerase, and one or more magnesium salts, comprising preparing an oligonucleotide primer-carrier nucleic acid admixture containing one or more amplification primers and carrier nucleic acid, and contacting the primer-carrier mixture with the target nucleic acid, polymerase, and magnesium salts.
In a fourth aspect, the invention is directed to a method for reducing the formation of primer-dimer or other non-specific nucleic acid amplification products in a reaction for the amplification of a target nucleic acid, where the amplification reaction mixture comprises one or more oligonucleotide amplification primers specific to the target, polymerase, and one or more magnesium salts, comprising preparing a primer-
Backus John W.
Preston Gregory M.
Darby & Darby
Horlick Kenneth R.
Ortho-Clinical Diagnostics, INC
Strzelecka Teresa
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