Self-primed amplification system

Details Self-primed amplification system Self-primed amplification system

1999-11-23

2001-03-27

Jones, W. Gary (Department: 1656)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091200, C536S024300, C536S024330, C536S025300

Reexamination Certificate

active

06207424

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATIONS
[Not Applicable]
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
[Not Applicable]
FIELD OF THE INVENTION
This invention relates to the field of copying a nucleic acid. An improved method of copying a nucleic acid with a self-priming primer is provided. Methods for using the self-priming primer to copy single and different target nucleic acids are described.
BACKGROUND OF THE INVENTION
Methods for copying nucleic acids provide useful tools for the detection of human pathogens, the detection of human genetic polymorphisms, molecular cloning, the detection of RNA and DNA sequences, sequencing of nucleic acids, and the like. In particular, the polymerase chain reaction (PCR) has become an important tool in the cloning of DNA sequences, forensics, paternity testing, pathogen identification, disease diagnosis, and other useful methods where the copying of a nucleic acid sequence is desired. See e.g.,
PCR Technology: Principles and Applications for DNA Amplification
(Erlich, ed., 1992);
PCR Protocols: A Guide to Methods and Applications
(Innis et al., eds, 1990).
PCR permits the copying of a target nucleic acid. Briefly, a target nucleic acid, e.g. DNA, is combined with a sense and antisense primers, dNTPs, DNA polymerase and other reaction components. See Innis et al. The sense primer can anneal to the antisense strand of the DNA target. The antisense primer can anneal to the sense strand of the DNA target, downstream of where the sense primer anneals to the DNA target. In the first round of amplification, the DNA polymerase extends the antisense and sense primers that are annealed to the target nucleic acid. The first strands are synthesized as long strands of indiscriminate length. In the second round of amplification, the antisense and sense primers anneal to the parent target nucleic acid and to the complementary sequences on the long strands. The DNA polymerase then extends the annealed primers to form strands of discrete length that are complementary to each other. The subsequent rounds serve to predominantly amplify the DNA molecules of the discrete length.
The sense and antisense primers, however, lack the ability to self-prime. That is, the primers are not able to generate an oligonucleotide from their own sequence during amplification that will lead to the desired product. Another drawback of traditional PCR is that artifacts can be generated from mis-priming and primer dimerization. Those artifacts can be exacerbated in traditional multiplex PCR. Multiple sets of primers increase the possibility of primer complementarity at the 3′-ends, leading to primer-dimer formation. These artifacts deplete the reaction of dNTPs and primers and outcompete the multiplex amplicons for DNA polymerase. Such artifacts can be reduced by careful primer design and the use of “hot start” PCR. See Chou, Q. et al (1992)
Nucleic Acids Research,
20: 1717-1723. It is increasingly difficult to eliminate all interactions that promote the mis-priming and primer dimerization, however, in a multiplex amplification as the reaction may contain many primers at high concentration.
Additionally, in a multiplex amplification, it is desirable that the amplification of different targets is quantitative, i.e., that the amplification accurately reflects the true ratio of target sequences in the sample. The data obtained using traditional multiplex PCR, however, is at best semi-quantitative. Amplification of sets of amplicons of varying lengths and GC-content may show preferential amplification of the shortest length and lowest GC-content amplicon. Further, differences in the yields of unequally amplified fragments are enhanced with each cycle. Multiplex PCR has been observed to suppress the amplification of one amplicon in preference for another amplicon. A number of factors are involved. For example, when a multiplex PCR involves different priming events for different target sequences, the relative efficiency of these events may vary for different targets. This can be due to the differences in thermodynamic structure stability and hybridization kinetics among the various primers used. Moreover, if the kinetics of product strand renaturation differ for different targets, the extent of competition with priming events will not be the same for all targets.
Accordingly, there is a need for compositions and methods that are less likely to produce variable and erroneous signals in multiplex assays. The present invention solves these problems and provides useful methods and compositions for carrying out a self-primed nucleic acid replication.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions for copying a target nucleic acid using a self-primed amplification system to copy a single or different target nucleic acid sequences.
In one aspect, the present invention provides for a composition of a self-priming nucleic acid polymerase (SNAP) primer for copying a target nucleic acid comprising four domains of nucleic acid sequences. The domains are aligned in the following order from 3′ to 5′: i. a sense, target-specific binding domain; ii. a non-target, antisense primer binding domain; iii. a non-target sense primer binding domain; iv. an antisense, target-specific binding domain. The sequence of domain iv, the antisense, target-specific binding domain complements a sense sequence of the target which lies upstream of the target sequence complementary to the sense, target-specific binding domain.
In another aspect, the present invention provides for a mixture of amplification primers composed of first and second primers for copying a target nucleic acid. The first primer is comprised of two domains of nucleic acid sequences in the following order from 3′ to 5′: i. a sense, target-specific binding domain; and ii. a non-target, antisense primer binding domain. The second primer is comprised of two domains of nucleic acid sequences in the following order from 3′ to 5′: i. a non-target sense primer binding domain; and ii. an antisense, target-specific binding domain. The sequence of the antisense, target-specific binding domain complements a sense sequence of the target nucleic acid which lies upstream of the target nucleic acid sequence complementary to the sense, target-specific binding sequence.
In another aspect, the present invention provides for a mixture of nucleic acid primers for copying a target nucleic acid comprising a primer mixture selected from a group consisting of three different members. The first member is comprised of a primer comprising two domains of nucleic acid sequences in the following order from 3′ to 5′: i. a sense, target-specific binding domain; ii. a non-target, antisense primer binding domain; and a primer having 3′ sequence identity to the sequence of the non-target, antisense primer binding domain. The second member is comprised of a primer comprising two domains of nucleic acid sequences in the following order from 3′ to 5′: i. a non-target sense primer binding domain; ii. an antisense, target-specific binding domain wherein the sequence of the domain complements a sense sequence of the target nucleic acid which lies upstream of the target nucleic acid sequence complementary to the sense, target-specific binding sequence; and a primer complementary to the non-target sense primer binding domain. The third member of the group is a combination of four primers making up the first and second members of the group.
In another aspect, the present invention provides for a method of copying a target nucleic acid. In the first step, the following components are combined in an aqueous solution: a target nucleic acid, a nucleic acid polymerase, a molar excess of dNTPs, a non-target antisense primer, a non-target sense-primer, and self-priming nucleic acid polymerase (SNAP) primers for copying a target nucleic acid. The SNAP primers comprise four domains of nucleic acid sequences in the following or

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