Molecular cloning using rolling circle amplification

Details Molecular cloning using rolling circle amplification Molecular cloning using rolling circle amplification

1999-09-15

2001-09-11

Whitenant, Ethan (Department: 1655)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C435S006120, C435S091100, C436S094000, C536S023100, C536S024300, C536S024330

Reexamination Certificate

active

06287824

ABSTRACT:

BACKGROUND OF THE INVENTION
The disclosed invention is generally in the field of molecular cloning and nucleic acid amplification, and specifically involves rolling circle replication of nucleic acid molecules inserted into circular vectors.
DNA molecular cloning is routinely carried out using plasmid, phage, or viral vectors that replicate inside cells. A method, in which individual DNA molecules are cloned in solution by serial dilution and subsequent PCR amplification from tubes containing single molecules has been described (Lukyanov et al.,
Nucleic Acid Research
24:2194-2195 (1996)). A method has also been described for cloning RNA populations derived from single RNA molecules in an immobilized medium (Chetverina and Chetverin,
Nucleic Acids Research
21:2349-2353 (1993)). While both of these methods allow in vitro cloning, neither is practical for high throughput cloning.
Velculescu et al.,
Science
270:484-487 (1995), have described a method for the quantitative cataloguing and comparison of expressed genes in normal, developmental, and disease states. The method, termed serial analysis of gene expression (SAGE), is based in the use of relatively short sequence tags for the unique identification of cDNAs derived from mRNA transcripts. While this method is very powerful, the study of low-abundance mRNAs can require several months of work in order to obtain sufficient sequence information for a complete SAGE analysis of one tissue sample. Thus, there is a need for a method to obtain the sequence of sequence tags more rapidly.
It is therefore an object of the present invention to provide a more efficient method of in vitro molecular cloning.
It is also an object of the present invention to provide vectors and kits useful for in vitro cloning.
It is also an object of the present invention to provide an automated method molecular cloning.
It is also an object of the present invention to provide a more efficient method of sequential analysis of gene expression.
BRIEF SUMMARY OF THE INVENTION
Disclosed are reagents and a method for efficient in vitro molecular cloning of nucleic acid molecules of interest. Because the method is entirely in vitro, it can be automated and scaled-up in ways that are not possible in cell-based molecular cloning. The method involves insertion of a nucleic acid molecule of interest in a linear vector to form a circular vector where one strand is continuous and the other strand is discontinuous. The continuous strand of the circular vector is then amplified by rolling circle replication, amplifying the inserted nucleic acid molecule in the process. The amplification is rapid and efficient since it involves a single, isothermic reaction that replicates the vector sequences exponentially. The amplification process is amenable to automation where multiple reactions are carried out simultaneously in a small area. The amplified nucleic acid can be used for any purpose and in any manner that nucleic acid cloned or amplified by known methods can be used. This includes sequencing, probing, restriction analysis, subcloning, transcription, hybridization or denaturation analysis, further amplified, and storage for future use or analysis.
The insertion reaction involves insertion of a double-stranded nucleic acid molecule into a double-stranded linear vector to produce a double-stranded circular vector. The use of circular vectors facilitates the selection of molecules that have successfully incorporated inserts. The amplification reaction involves rolling circle replication of a single-stranded circular nucleic acid molecule.
A key feature of the method, which facilitates double-stranded insertion followed by single-stranded amplification, is formation of the circular vector in such a way that one of its strands is a closed circular strand (that is, continuous) while the other strand is not a closed circular strand (that is, it has a nick, a gap, an overlap, or is otherwise discontinuous). This feature is most useful, and most effectively accomplished, when, by operation of the method, the closed strand and the open strand are predetermined; that is, when a particular strand of the vector is selectively left discontinuous.
With rolling circle replication, amplification takes place not in cycles, but in a continuous, isothermal replication. This makes amplification less complicated and much more consistent in output. A single round of rolling circle replication results in a large amplification of the circular vector, orders of magnitude greater than a single cycle of PCR replication and other amplification techniques in which each cycle is limited to a doubling of the number of copies of a target sequence.
Following amplification, the amplified nucleic acid can be used for any purpose. Numerous methods for the use and manipulation of cloned or isolated nucleic acid are known and can be applied to nucleic acid amplified in the present method. For example, the nucleic acid can be sequenced, probed, subjected to restriction analysis, subcloned, transcribed, subjected to hybridization or denaturation analysis, further amplified, or stored. Diagnostic methods, such as sequencing and probing for specific sequences, are preferred.
Libraries of cloned nucleic acids formed by the disclosed method can be screened using any of the methods used for screening conventional libraries. For example, cDNA libraries made using the disclosed method can be analyzed using conventional screens. Libraries can also be used for in situ transcription to generate RNA colonies, which can then be analyzed (in situ or in replicas) by appropriate screens, such as aptamer screens or ribozyme activity screens. Libraries can also be screened by in situ translation on array replicas (see, for example, Saris et al.,
Nucleic Acids Res.
10:4831-4843 (1982)). Libraries can also be screened by in situ coupled transcription-translation systems, and subsequent catalytic activity assays for the analysis of mutagenized enzymes. Libraries can be screened and cataloged by sequencing and use of the data for the analysis of cDNA abundancies, which is useful for RNA profiling and serial analysis of gene expression (SAGE; Velculescu et al.,
Science
270:484-487 (1995)).


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