Nucleic acid amplification using modular branched primers

Details Nucleic acid amplification using modular branched primers Nucleic acid amplification using modular branched primers

1997-05-06

2001-03-06

Whisenaut, Ethan (Department: 1655)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C435S006120, C536S023100, C536S024300

Reexamination Certificate

active

06197556

ABSTRACT:

The invention provides methods and compositions that eliminate the need for custom synthesis of primers used in methods of amplifying nucleic acid segments such as the Polymerase Chain Reactions (PCR). Instead of being custom-synthesized, a primer is replaced by a combination of several branched and/or covered oligonucleotide modules selected from a pre-synthesized library. A modular combination of just a few oligonucleotides essentially mimics the performance of a conventional, custom-made primer by matching a sequence of a priming site in a template.
There are growing needs to perform hundreds and thousands and in the near future—tens of thousands, and perhaps millions of different amplification (e.g. PCR) reactions in areas as genome mapping, biomedical research and clinical diagnostics. In genome mapping, the locations of thousands of markers are being determined relative to each other and to such clones as YACs, cosmids, BACs and PACs. These locations are continuously updated by performing many different PCR reactions, each corresponding to a different marker, because the number of available markers and clones is growing rapidly. In biomedical research, with the explosive growth of the number of known genes related to various diseases, the need to screen a large number of mutations in many genes simultaneously will very soon require prohibitively large numbers of different PCR reactions to be performed for determining the effect of the various mutations on the diseases. With the number of fully sequenced genomes growing rapidly and including human sequences, simultaneous comprehensive polymorphism tests of thousands of genes is likely. Large scale screening will then be required for each individual to test predisposition for various genetic conditions.
One of the limitations of the present methods is the need to prepare large numbers of customized primers. Rather than synthesizing primers specifically for each reaction, a presynthesized library could be made. However, maintenance and management of a library that contains many thousands of customized primers is a bottleneck, for example in genome mapping. About a million primers will be required in the near future for genome-wide mutation screening. Management of such a huge library is very difficult. One alternative, cheap de novo synthesis of primers each time they are needed, may in the future be viable for small number of primers, at best—thousands of primers at a time. For much larger numbers of primers, this alternative is prohibitively expensive. Manual handling of great numbers of PCR reactions is exceedingly difficult. Dedicated robots are already under development for this purpose. In this instrumentation development, the main outstanding problems are synthesis, storage and handling of a large number of different PCR primers.
Almost a million-fold discrepancy between the scale of primer synthesis (1.0 &mgr;mol) and the amount of primer required for a conventional PCR reaction (2.0 &mgr;mol) could be alleviated using a pre-synthesized library of possible primer sequences. Such a library could be aliquoted into thousands of copies for individual users, thus dramatically slashing the expense per reaction and making the primers instantly available. However the number of samples (sequences) in such a library would be too large to be practical. For example, even the shortest primer expected to be unique in a plasmid-sized template, a nonamer, would require a quarter of a million sequences to be prepared, each in a separate receptacle.
New approaches to nucleic acid amplification are needed that are economical and can be scaled up and automated.
SUMMARY OF THE INVENTION
The methods and compositions of the present invention eliminate the need for custom primer synthesis in methods to amplify nucleic acid segments, e.g., the polymerase chain reaction (PCR). One aspect of the invention makes it possible to instantly assemble a primer of a given sequence using a pre-synthesized, modular, oligonucleotide library. Modular primers may be branched or covered. Branched primers have a three dimensional structure that is basically a three-way junction when annealed to a template. Covered primers are similar except for the absence of one portion of the junction. The stem portions of branched primers are constant and bind portions of variable modules together to give specificity to the initial priming (extension) yet allow amplification using conventional primers to proceed.
The invention is directed to methods and compositions for amplifying a segment of a nucleic acid template, in particular by a polymerase chain reaction. The method includes annealing the template to a first branched primer which includes both front and back oligonucleotide modules. “Front” refers herein to the 3′ extending (downstream) sequence and “back” refers to the 5′ end (upstream), both terms are in reference to the direction of extension of a sequence by the polymerase. Each oligonucleotide module sequence has a nucleotide sequence designated the stem segment. The stem segment sequences of the front and back oligonucleotide modules are complements of each other, and therefore are capable of annealing to each other. The stem segments when annealed form the stem of a branched primer.
The two oligonucleotide modules (front and back) also have an “arm segment” which is complementary to a nucleotide sequence site in a template. A “template” is a nucleic acid segment, a part of which is to be amplified. The sites to which the two arm segments are complementary are sufficiently close to each other in the template so that the first branched primer forms a 3-way junction when annealed to the template. The arm of the front oligonucleotide module of the first branched primer is extended on the template by a polymerase enzyme to form a first initial extension strand. The first initial extension strand is then annealed to a reverse primer which may be either branched (a second branched primer) or not and which is extended on the first initial extension strand by a polymerase enzyme to form a second initial extension strand. The second initial extension strand is amplified by using amplification primers that include a reverse primer and/or at least one primer homologous to the stem sequence of the first and/or second branched primer. The arm of each oligonucleotide module sequence preferably contains at least one artificial base to reduce steric hindrance that may be caused by proximity of the stem to the extension point and/or to enhance the annealing stability. An intercalator group is optionally linked to an arm to stabilize the annealing of the arm to the template. Two examples of intercalators to stabilize the annealing are 1) acridine, and 2) 2-methoxy6-chloro-9-aminoacridine.
Optionally, at least one of the back modules of the amplification primers is a 3′ protruding sequence, i.e., when annealed to its priming site, the primer covers one or more extra bases in this site as compared to the front module. The protruding amplification primers preferably comprise artificial non-discriminating bases in order to reduce the number of possible sequences of these primers (the size of primer library.)
The invention relates a method for amplifying a segment of a nucleic acid template, said method comprising:
a. annealing the template to a first branched primer, said primer comprising a front and a back oligonucleotide module, wherein (i) the front oligonucleotide module comprises a stem segment, and (ii) wherein said stem segment is complementary to a stem segment of the back oligonucleotide module, and (iii) wherein said oligonucleotide modules anneal to each other by means of said stem segments; (iv) wherein each of said oligonucleotide modules also comprises an arm segment, and (v) wherein said segment is complementary to a site in the template, and (vi) wherein the two sites are sufficiently close to each other in the template so that said first branched primer forms a 3-way junction when annealed to the template;
b. extending the arm of the front mo

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