Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-05-12
2002-10-29
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C435S091200, C536S023100, C536S063000, C536S024330, C536S025300, C530S334000
Reexamination Certificate
active
06472184
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for producing nucleic acid polymers.
BACKGROUND OF THE INVENTION
The increasing use of recombinant genes in genetic engineering and biotechnology and in the medical analytical field has created a great demand for methods of the “de novo” synthesis of long nucleic acid chains. In many cases the synthetic production of arbitrarily chosen nucleic acid sequences can be a time-saving alternative to troublesome cloning and modifying methods. A routine synthesis of long nucleic acid chains can also offer decisive advantages in the case of “drug design”, e.g. in the production of “custom-made” antibodies, inhibitor/activator molecules, ribozymes, or in DNA chip technology. Furthermore, purposefully modified nucleotides, e.g. labeled by (fluorescent) dyes or enzymes, could be incorporated into a nucleic acid chain in this way. Apart from the examples given here, there are many further possible applications for purposefully produced nucleic acid polymers.
In the simplest variant of conventional gene synthesis, i.e. direct cloning, two long oligonucleotides which are fully complementary to each other are hybridized with one another. On account of the present technical limitation in the production of oligonucleotides to a length of 150 to 200 bases, the size of the resultant hybridization products is also limited to such a range of length.
A further method for producing long nucleic acid polymers is the so-called fill-in method in which two single-stranded nucleic acid chains are hybridized with each other, and protruding ends are filled with the help of DNA polymerases so that the double-stranded product is longer than the oligonucleotides used. However, even with the fill-in method, it is not possible to produce a product that is longer than the sum of the nucleic acid chains used.
In the “shot-gun gene synthesis” complementary single-stranded oligonucleotides are directly transfected into cells together with an expression vector which has been opened by restriction enzymes, and a circularized product can here only be obtained if all of the partial sequences are ligated by the enzyme machinery of the host organism in a suitable way. The efficiency of successful ligations in this method is in general very limited, in particular when many oligonucleotides are used for gene synthesis.
In 1972 Khorana developed a method named after him, in which several chemically synthetized oligonucleotides of an average length of 15 nucleotides, which in a suitable arrangement are overlapping without any gaps, were enzymatically joined by polynucleotide ligase to obtain a double strand (Khorana, H. G. et al., J. Mol. Biol. 27, 209-17, 1972, and follow-up publications; Khorana, H. G. et al., J. Biol. Chem. 251, 3(10), 565-70, 1976 and follow-up publications). Sequential ligation of a few (4-8) oligonucleotides for obtaining longer intermediate products, purification of the intermediate products and subsequent ligation of the intermediate products with one another resulted in the synthesis of double-stranded nucleic acid chains with a length of 514 base pairs (Edge, M. D. et al., Nature 292, 756-62, 1981), later with a length of up to about 1000 bp, which could subsequently be cloned in bacterial expression vectors.
These methods have several decisive drawbacks:
1.) After each ligation step the products or intermediate products had to be purified by separation on a polyacrylamide gel (PAA gel) to eliminate undesired by-products of the ligation reaction. Such a time-intensive working step requires great efforts with respect to personnel and costs.
2.) During elution of the intermediate and final products from the PAA gel considerable losses in yield had to be accepted.
3.) The so far longest product that could be produced with the help of said technique had a length of about 1000 base pairs. Since most of the eukaryotic and prokaryotic genes have a coding sequence of 300 to 3000 base pairs on the average, such a length is not sufficient for most applications.
4.) For the necessary purification via PAA gels the nucleotides were normally radioactively labeled in the method described by Khorana to be able to identify the bands of the desired products in the gel. The use of highly radioactive
32
P labels constitutes a potential risk which could not be avoided in said method.
OBJECT OF THE INVENTION
It is therefore the object of the present invention to provide an uncomplicated, reliable and inexpensive method for the synthesis of nucleic acid polymers of a length of more than 1000 bases in one step, wherein the above-mentioned drawbacks can be overcome.
DETAILED DESCRIPTION OF THE INVENTION
Said object is achieved by a method for producing a nucleic acid polymer, comprising the following steps:
a) providing 2 or more linkable oligonucleotides which in a continuous arrangement and after linkage can form a primary strand, and one or more non-linkable oligonucleotides, each of the non-linkable oligonucleotides comprising two adjoining regions the first of which is complementary to the 3′ end of a linkable oligonucleotide and the second of which is complementary to the 5′ end of a further linkable oligonucleotide,
b) hybridizing oligonucleotides for the primary strand with the complementary regions of the non-linkable oligonucleotides, and
c) linking the oligonucleotides of the primary strand.
The method according to the invention is schematically shown in
FIGS. 1 and 2
. In contrast to the Khorana method, it offers the decisive advantage that the synthesis of a single-stranded nucleic acid polymer can be carried out in a single reaction batch. All of the linkable and non-linkable oligonucleotides that are required for the synthesis of the primary strand are here used at the same time; the addition of a linker yields a primary strand of covalently linked oligonucleotides which can reach a length of more than 1000, e.g. 1500, bases.
In preferred embodiments steps (b) and (c) are repeated several times, and the oligonucleotides which were previously hybridized with one another are separated from each other prior to each repetition of said two steps, i.e. the double strand previously formed by hybridization is denatured. Denaturation can be carried out by increasing the temperature or by increasing the pH in the manner known to one skilled in the art. The repeated denaturation and renaturation with subsequent linking considerably improves the yield in primary strand which, otherwise, is e.g. impaired by chain terminations which are the result of the incorporation of incompletely phosphorylated primary strand oligonucleotides. The influence of such chain terminations on the total yield is minimized by repeating steps (b) and (c).
According to the invention steps (b) and (c) are carried out not only once, but several times. In a preferred embodiment, they are repeated 1 to 8 times, particularly preferably 3 to 5 times.
The number of the oligonucleotides used can vary between 2 linkable oligonucleotides and 150 linkable oligonucleotides. The number of the non-linkable oligonucleotides is always equal to the number of the linkable oligonucleotides, by one higher or by one lower, i.e. at least one. Between 5 and 100 linkable oligonucleotides are preferably used; particularly preferably between 10 and 50 linkable oligonucleotides.
The linking of the linkable oligonucleotides of the primary strand may comprise various reactions. In this instance linking means e.g. a reaction of an enzymatic, chemical or also photochemical kind. For instance in the case of enzymatic linking (ligation), T4 DNA ligase is e.g. used as the linker. In the preferred embodiment, use is made of a thermostable ligase, e.g. Pfu ligase (Stratagene) which offers the advantage that in the case of repeated cycles of denaturation by way of temperature increase, hybridization and ligation of the oligonucleotides, no new enzyme has to be added whereas this is the case when thermolabile DNA ligases, such as T4 DNA ligase, are used.
Furthermore, the use of said thermostable ligase m
Chakrabarti Arun
Darby & Darby
Fredman Jeffrey
LandOfFree
Method for producing nucleic acid polymers does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for producing nucleic acid polymers, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for producing nucleic acid polymers will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2973002