Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-06-28
2001-11-27
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091200
Reexamination Certificate
active
06323009
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to processes for establishing multiple replication forks in rolling circle amplification so as to provide enhanced yields of amplification products, with quantitative advantages over previous rolling circle methods.
BACKGROUND OF THE INVENTION
A means of amplifying circular target DNA molecules is of value because such amplified DNA is frequently used in subsequent methods including DNA sequencing, cloning, mapping, genotyping, generation of probes, and diagnostic identification.
Heretofore, several useful methods were developed that permit amplification of nucleic acids. Most were designed around the amplification of selected DNA targets and/or probes, including the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and amplification with Q&bgr; replicase (Birkenmeyer and Mushahwar,
J. Virological Methods,
35:117-126 (1991); Landegren,
Trends Genetics,
9:199-202 (1993)).
In addition, several methods have been employed to amplify circular DNA molecules such as plasmids or DNA from bacteriophage such as M13. One has been propagation of these molecules in suitable host strains of
E. coli,
followed by isolation of the DNA by well-established protocols (Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning, A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). PCR has also been a frequently used method to amplify defined sequences in DNA targets such as plasmids and DNA from bacteriophage such as M13 (PCR Protocols, 1990, Ed. M. A. Innis, D. H. Gelfand, J. J. Sninsky, Academic Press, San Diego.) Some of these methods suffer from being laborious, expensive, time-consuming, inefficient, and lacking in sensitivity.
As an improvement on these methods, linear rolling circle amplification (LRCA) uses a primer annealed to a circular target DNA molecule and DNA polymerase is added. The amplification target circle (ATC) forms a template on which new DNA is made, thereby extending the primer sequence as a continuous sequence of repeated sequences complementary to the circle but generating only about several thousand copies per hour. An improvement on LRCA is the use of exponential RCA (ERCA), with additional primers that anneal to the replicated complementary sequences to provide new centers of amplification, thereby providing exponential kinetics and increased amplification. Exponential rolling circle amplification (ERCA) employs a cascade of strand displacement reactions, also referred to as HRCA (Lizardi, P. M. et al.
Nature Genetics,
19, 225-231 (1998)). However, ERCA is limited to the use of just a single primer P1 annealed to the circular DNA target molecule, to the need to know the specific DNA sequence for the primer P1, and for the need of the circular DNA target molecule to be a single-stranded DNA circle.
The methods of the present invention (referred to herein as Multiply-Primed Rolling Circle Amplification—MPRCA) avoid such disadvantages by employing procedures that improve on the sensitivity of linear rolling circle amplification by using multiple primers for the amplification of individual target circles. The present invention has the advantage of generating multiple tandem-sequence DNA (TS-DNA) copies from each circular target DNA molecule. In addition, MPRCA has the advantages that in some embodiments the sequence of the circular target DNA molecule may be unknown while the circular target DNA molecule may be single-stranded (ssDNA) or double-stranded (dsDNA or duplex DNA). Another advantage of some embodiments of the present invention is that the amplification of single-stranded or double-stranded circular target DNA molecules may be carried out isothermally and/or at ambient temperatures. Other advantages include being highly useful in new applications of rolling circle amplification, low cost, sensitivity to low concentration of target circle, flexibility, especially in the use of detection reagents, and low risk of contamination.
In some embodiments of the present invention, procedures are employed that improve on the yield of amplified product DNA by using multiple primers that are resistant to degradation by exonuclease activity that may be present in the reaction. This has the advantage of permitting the primers to persist in reactions that contain an exonuclease activity and that may be carried out for long incubation periods. The persistence if primers allows new priming events to occur for the entire incubation time of the reaction, which is one of the hallmarks of ERCA and has the advantage of increasing the yield of amplified DNA.
The methods of the present invention allow for the first time “in vitro cloning”, i.e. without the need for cloning into an organism, of known or unknown target DNAs enclosed in circles. A padlock probe may be used to copy the target sequence into a circle by the gap fill-in method (Lizardi, P. M. et al.
Nature Genetics,
19,225-231 (1998)). Alternatively, target sequences can be copied or inserted into circular ssDNA or dsDNA by many other commonly used methods. The RCA amplification overcomes the need to generate amplified yields of the DNA by cloning in organisms.
One application envisioned is the targeted capture of known sequences from genomic or other complex DNAs. A second application is RCA of circles generated in a whole genome amplification method. Whole genome amplification involves randomly primed or specifically primed generation of a subset of genomic, cDNA or other complex DNA. Methods well known in the art can be used to circularize the products of whole genome amplification. Padlocks could also generate the circular targets. These circles would then constitute substrates for the targeted amplification of the present invention. Regardless of the means used to generate the circular products of whole genome amplification, the random priming RCA of the present invention would allow the selective amplification of the circles over the background of linear DNAs without the need for knowing sequences. Similarly, the circular DNA could contain known vector or target sequences that would allow use of specific primer sequences for multiple primer RCA.
The methods of the present invention are an improvement over LRCA in allowing increased rate of synthesis and yield. This results from the multiple primer sites for DNA polymerase extension. Random primer RCA also has the benefit of generating double stranded products. This is because the linear ssDNA products generated by copying of the circular template will themselves be converted to duplex form by random priming of DNA synthesis. Double stranded DNA product is advantageous in allowing for DNA sequencing of either strand and for restriction endonuclease digestion and other methods used in cloning, labeling, and detection.
It is also expected that strand-displacement DNA synthesis may occur during random priming RCA resulting in an exponential amplification. This is an improvement over conventional ERCA, also termed HRCA (Lizardi et al. (1998)) in allowing for the ability to exponentially amplify very large targets enclosed in circles. The amplification of large circular DNA, including bacterial artificial chromosomes (BACs), has been reduced to practice in the present invention. In practice, conventional ERCA has been limited to use of small circles of less than 200 nucleotides length.
Methods have published for whole genome amplification using degenerate primers (Cheung, V. G. and Nelson, S. F.
Proc. Natl. Acad. Sci. USA,
93, 14676-14679 (1996) and random primers (Zhang, L. et al.,
Proc. Natl. Acad. Sci. USA,
89, 5847-5851 (1992) where a subset of a complex mixture of targets such as genomic DNA is amplified. Reduction of complexity is an objective of these methods. A further advantage of the method of the present invention is that, as an RCA reaction, it selectively amplifies circular DNA target molecules without the need
Dean Frank B.
Lasken Roger S.
Nelson John
Grant Alan J.
Horlick Kenneth R.
Molecular Staging Inc.
Olstein Elliot M.
Strzelecka Teresa
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