Linked linear amplification of nucleic acids

Details Linked linear amplification of nucleic acids Linked linear amplification of nucleic acids

1999-01-11

2002-01-01

Houtteman, Scott W. (Department: 1634)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C435S006120

Reexamination Certificate

active

06335184

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to the in vitro replication of nucleic acids. More specifically, the invention relates to a process for replicating a nucleic acid sequence of interest, with large quantities of the desired sequence ultimately resulting from the linkage of primer extension reactions wherein the sequence of interest accumulates in a mathematically linear fashion.
2. Brief Description of the Background Art
The extensive replication of nucleic acids, today known as (and referred to herein as) nucleic acid “amplification,” finds wide utility, both practical and theoretical, in a variety of contexts. H. G. Khorana and his co-workers first proposed the use of an in vitro DNA amplification process to increase available amounts of double-stranded DNA (partial sequences of the gene for the major yeast alanine t-RNA) that had been created by the enzymatic ligation of synthetic DNA's. See K. Kleppe et al.; J. Mol. Biol. 56:341-361 (1971). Later, in vitro amplification was applied to the amplification of genomic DNA (Saiki et al., Science 230:1350-1354 (1985)) as the technique now known as the polymerase chain reaction or “PCR.” Through the wide availability of synthetic oligonucleotide primers, thermostable DNA polymerases and automated temperature cycling apparatus, PCR became a widely-utilized tool of the molecular biologist.
The PCR process is referred to in the literature as an “exponential amplification” process. In each round or “cycle” of primer extension, a primer binding site for the other primer is synthesized. Thus, each of the synthetic DNA molecules produced in any of the previous cycles is available to serve as a template for primer-dependent replication. This aspect of the process, coupled with the presence of a sufficiently large number of primer molecules, results in synthetic DNA accumulating in a mathematically exponential manner as the reaction proceeds.
Although PCR has proven to be a valuable technique for the molecular biologist, and has been used extensively in the fields of human genetic research, diagnostics and forensic science, and even in the detection of antibodies, disadvantages nevertheless have been recognized. The PCR process can be difficult to quantify accurately, mainly because the amplification products increase exponentially with each round of amplification. The products of PCR, namely, double-stranded DNA molecules, are difficult to analyze or sequence per se. Strand separation typically must be carried out prior to sequencing or other downstream processes that require single stranded nucleic acids, such as hybridization to a probe capable of detecting the sequence of interest.
The PCR process also has proven to be quite susceptible to contamination generated through the transfer of previously amplified DNA sequences into a new reaction. This problem appears to be caused by the facts that (1) very large amounts of DNA are generated in any given reaction cycle and (2) the process uses all product DNA strands as templates in subsequent cycles. Even minute quantities of contaminating DNA can be exponentially amplified and lead to erroneous results. See Kwok and Higuchi, Nature 339:237-238 (1989). Various methods to reduce such contamination have been reported in the literature (e.g. chemical decontamination, physical treatment, enzyme treatment and utilizing closed systems), as these contamination problems are widely recognized. See, John B. Findlay, “Development of PCR for in vitro Diagnostics,” presented at “Genetic Recognition,” Nov. 20, 1992, San Diego, Calif.
There has remained a need for new nucleic acid (DNA) amplification methods that provide large amounts of DNA, and that selectively amplify only a specific sequence of interest, but which avoid the problems now associated with the “PCR” reaction. Specifically, there has remained a need for nucleic acid amplification methods that ultimately produce large amounts of a nucleic acid molecule of interest, or large amounts of a molecule containing a nucleic acid sequence of interest, but are relatively insensitive to the presence of contaminating nucleic acids. There has also remained a need for nucleic acid amplification methods that generate single-stranded products.
SUMMARY OF THE INVENTION
The foregoing and other needs are met by the present invention, which in one aspect provides a process for amplifying a specific nucleic acid sequence of interest within complementary nucleic acid strands contained in a sample, the process including the steps of:
(a) contacting the strands with a primer that contains a non-replicable element, under conditions such that first generation primer extension products are synthesized using said strands as templates, and wherein the primer for a strand is selected such that a first generation primer extension product synthesized thereon, when separated from the strand, can serve as a template for synthesis of a second generation primer extension product of the primer for the complement of the strand;
(b) separating the first generation primer extension products from their templates to produce single-stranded molecules; and
(c) treating the first generation primer extension products with the primers of step (a) under conditions such that second generation primer extension products are synthesized using the first generation primer extension products as templates; wherein the second generation primer extension products contain at least a portion of the nucleic acid sequence of interest and cannot serve as templates for the synthesis of extension products of the primers which were extended to synthesize their templates.
In other aspects of the invention, the products of step (c) are separated to produce single-stranded molecules, and the entire process is repeated at least once. Step (c) preferably is repeated many times, with the process being carried out in an automated fashion under the control of a programmable thermal cycling apparatus.
Following the accumulation of second generation primer extension products, each of which is incapable of serving as a template for the primer extended to prepare its first generation template, a new set of primers that contain non-replicable elements can be employed. The new set of primers advantageously bind to the second generation synthetic products, bounding the sequence of interest to be amplified. The linear replication process is again carried out through a number of cycles. Such “linking together” of multi-cycle primer extension reactions ultimately results in thousand-fold or million- fold amplification of the original nucleic acid sequence of interest. Thus, the present process is deemed “linked linear amplification” or “LLA.”
In another aspect of the invention, multiple (nested) sets of primers containing non-replicable elements can be provided in a single amplification reaction mixture. The sets are selected so as to be capable of binding to their respective templates under decreasingly stringent conditions. Alternatively, as in Examples 8-11, the LLA reaction can be performed using a nested set of primers under a single stringency condition under which all of the primers in the set are capable of simultaneously binding to their respective complementary sites. Thus, all the components necessary to carry out several linked linear amplifications can be provided in a single reaction mixture.
In yet another aspect of the invention, allele- specific nucleic acid replication is carried out according to the present invention with the use of primers directed to specific polymorphic sites on the template that are known to be indicative of a genetic disease or disorder, such as sickle cell disease. The allele-specific primers, containing non-replicable elements, are designed so that they prime nucleic acid synthesis of only those templates containing the desired allele.
The synthetic nucleic acid molecules resulting from the present process can be used in the diagnosis of genetic disorders or diseases, as reagents in further techniques such as gene cloning, for foren

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