Method for dna amplification based on the origins of replication of the bacteriophage p29 and associated nucleotide sequences
AssigneeConsejo Superior De Envestigaciones Cientificas (Csic)
InventorsMargarita Salas Falgueras, Mario Mencia Caballerro, Miguel De Vega Jose, Jose M. Lazaro Bolos, Pablo Gella Montero
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Abstract
The present invention refers to a DNA amplification method based on the origins of replication of bacteriophage φ29, and to the genic constructs, vectors and oligonucleotides that can be used in the method for amplifying an exogenous sequence of interest.
Claims33
A genic construct adapted for the introduction of an exogenous nucleotide sequence, and which consists of at least a nucleotide sequence with the following three elements in the stated order:
- a) at least a nucleotide sequence comprising a replication origin of the DNA of bacteriophage φ29, with a terminal extremity followed by:
- b) at least one cleavage point of the nucleotide sequence,
- c) at least a nucleotide sequence comprising a replication origin of the DNA of bacteriophage φ29 with an orientation opposite that of the nucleotide sequence in (a).
The genic construct according to the above claim, where the nucleotide sequences of the three elements are overlapped.
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A DNA amplification method comprising at least the following steps:
- a) obtainment of a linear DNA molecule comprising the DNA sequence destined for amplification and flanked on both extremities by:
- i) the sequence of the left replication origin of the DNA of bacteriophage φ29, the terminal extremity of this origin being located in the linear DNA extremity;
- or by
- ii) the sequence of the right replication origin of the DNA of bacteriophage φ29, the terminal extremity of this origin being located in the linear DNA extremity;
- or by
- iii) the sequence of the right replication origin of the DNA of bacteriophage φ29 on one side and of the left replication origin on the other—the terminal extremity of these origins being located in the two extremities of the linear DNA;
- b) amplification of the linear DNA sequence obtained in step (a).
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A kit for the amplification of an exogenous nucleotide sequence, consisting of at least a genic construct adapted for the introduction of an exogenous nucleotide sequence, and which consists of at least a nucleotide sequence with the following three elements in the stated order:
- a) at least a nucleotide sequence comprising a replication origin of the DNA of bacteriophage φ29, with a terminal extremity followed by:
- b) at least one cleavage point of the nucleotide sequence,
- c) at least a nucleotide sequence comprising a replication origin of the DNA of bacteriophage φ29 with an orientation opposite that of the nucleotide sequence in (a);
- and a suitable buffer.
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Dependencies are inferred from the claim text.