Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-12-29
2001-07-24
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C536S023100, C536S024100
Reexamination Certificate
active
06265159
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method for generating DNA nested deletions by in vitro reactions.
The present invention also relates to a kit for use in said method for generating DNA nested deletions.
In genetic engineering technology, a desired DNA fragment is often manipulated by being insertedinto a vector. In such a case, it may be necessary to prepare the DNA insert in a length suitable for maniuplation.
For example, it is important to insert a DNA fragment of a suitable length into a vector, particularly in DNA sequencing techniques which can determine only a limited length of sequence at once. More specifically, the current DNA sequencing techniques can determine a sequence of at most about 1000 bases, usually only 300 to 400 bases at once. Therefore, longer fragments are sequenced by 1) subcloning, 2) primer walking, and 3) nested deletion or other methods. Among them, the nested deletion method is a technique which generates a number of nested deletions from a fixed site and it is more promising than the former two methods because a variety of deletion products can be conveniently obtained, as well as for other reasons. The nested deletion method hitherto known is performed in vitro by using exonuclease such as ExoIII or in vivo by using the terminal repeat of a transposon and transposase.
Transposons are a kind of movable genetic element and form a genetic unit which moves (transposes) from a portion of chromosomal DNA, plasmid DNA or viral DNA to another portion of the same or different DNA. They are widely distributed in bacteria, yeasts, maize, Drosophila, etc. The DNA site (target) to which they transpose is not fixed specifically, and it is presumed that they are able to transpose to any DNA site. Transposons are structurally characterized by an inverted repeat (IR) or a direct repeat at each end where recombination always occurs, indicating that this repeat has an important role in transposition. Transposons typically contain a gene responsible for the functions required for transposition or the expression of these functions.
Some transposons such as Tn3 and Tn1000 are known to function not only to bring about transposition of a gene but also to delete adjacent genes. Moreover, it was found that a mutation in the gene controlling transposition (tnpR) in a transposon increases the frequency of deletions as well as transposition reactions (Yoshinobu SUGINO and Hitoshi KAWASHIMA, Jpn. J. Genet. (1983) 58, pp. 79-93). A system for generating deletions in vivo has been developed (Sugino, Y. and Morita, M. 1994, Gene, 148, 169-170; and Wang, G. et al., Proc. Natl. Acad. Sci. USA (1993) 90, 7874-7878). This system is designed to insert a desired cloned DNA fragment into a vector having the terminal repeat of a transposon and transform said vector into
E. coli
which over expresses transposase to generate deletions from the terminal repeat of the transposon in
E. coli
. This system is commercially available as, for example, DELETION FACTORY® System from LIFE TECHNOLOGIES.
Although the conventional nested deletion methods are useful as described above, they involve problems due to in vivo reactions, e.g. they necessarily require a time for incubation and the desired gene may be denatured during reactions, etc. On the other hand, a transposon has been successfully transposed in vitro from a plasmid into &lgr; DNA (Ichikawa, H. and Ohtubo, E. 1990, J. Biol. Chem., 265,18829-18832). However, any method for generating nested deletions in vitro has not been known prior to the present invention, despite of a great need therefor.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method for producing nested deletions in vitro in a desired DNA, comprising:
1) preparing a vector containing a DNA fragment in which nested deletions are to be generated, and the terminal repeat of a transposon;
2) incubating said vector in vitro with transposase and a DNA replication system;
3) obtaining said vector incorporating said DNA fragment with nested deletions as a reaction product; and optionally,
4) transforming a host cell with said reaction product and growing it; and
5) recovering a vector incorporating said DNA fragment with nested deletions from the grown host cell.
Another object of the present invention is to provide kit for use in said method for generating DNA nested deletions, comprising transposase and a DNA replication system stored in a suitable container.
REFERENCES:
patent: 5645991 (1997-07-01), Berg et al.
patent: 5728551 (1998-03-01), Devine et al.
Morita et al. An in vitro system involving Tn3 transposase that catalyzes the formation of DNA deletions. Proc. Japan Acad. Vol. 65, Ser B, (1):1-4, Dec. 1989.*
Morita et al. Nested deletions from a fixed site as an aid to nucleotide sequencing: an in vitro system using Tn3 trasposase. DNA Research. vol. 3:431-433, Dec. 1996.*
LaBanca et al. Restriction map of a 35-kb HLA fragment constructed by nested deletion ‘drop-out’ mapping. Gene 164:335-339, Nov. 1, 1995.*
Sugino et al Jpn. J. Genet. (1983) 58, pp. 79-93 Deletion caused by Tn3: Correlation between deletion and transposition.
Sugino et al Gene. 148 (1994) 169-170 A new DNA cloning/sequencing vector with a built-in mechanism for generation of nested deletions using transposon Tn3*.
Wang et al Proc. Natl. Acad. Sci 90 7874-7878(Aug. 1993)pDual: A transposon-based cosmid cloning vector for generating nested deletions and DNA sequencing templates in vivo.
Ichikawa et al J. of Biological Chemistry 265, 31 pp. 18829-18832, 1990 In Vitro Transposition of Transposon Tn3*.
Morita et al J. Biochem 101 1253-1264 (1987) pp. 1253-1263 Overproduction and Purification of the Tn3 Transposase.
Matuo Yushi
Morita Masayuki
Sugino Yoshinobu
Uchida Kohji
Nixon & Vanderhye P.C.
Oriental Yeast Co. Ltd.
Sandals William
Schwartzman Robert A.
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