Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-06-28
2002-07-16
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091200, C435S440000, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06420144
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to a method for automated in vitro molecular cloning and amplification. More particularly, the invention relates to a process for the cell-free automated molecular cloning of a donor DNA which has been inserted into any nucleotide position in a recipient DNA, and amplifying the recombinant closed circular DNA clone in a single process.
Conventional methods for molecular cloning require multiple separate processes: first, donor and recipient DNA have to be digested by the proper restrictive enzymes; the digested DNA fragments have to be isolated and then subjected to a ligation process in order to insert the donor DNA into the recipient DNA; the resulting donor-recipient DNA is then used to transform the proper host cell to be able to amplify the recombinant donor-recipient DNA; and finally a selection process is performed to select the desired molecular clone with the correct insertion orientation. These conventional methods typically require several days to weeks of labor intensive manipulation in order to obtain and identify the desired molecular clone. Furthermore, cloning by conventional methods is limited by the availability of restriction sites on the recipient and/or donor DNAs. In order to obtain the desired clone, site-directed mutagenesis is often required to introduce restriction sites into the recipient and/or donor DNAs. This also makes the cloning process very labor-intensive and time-consuming.
Polymerase chain reaction (PCR) is a powerful method for the rapid and exponential amplification of target nucleic acids. E.g., U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporated by reference. PCR has facilitated the development of gene characterization and molecular cloning technologies including direct sequencing of PCR-amplified DNA, the determination of allelic variation, and the detection of infectious and genetic disorders. PCR is performed by repeated cycles of heat denaturation of a DNA template containing the target sequence, annealing of opposing primers to the complementary DNA strands, and extension of the annealed primers with a DNA polymerase. Multiple PCR cycles result in the exponential amplification of the nucleotide sequence delineated by the flanking amplification primers.
An important modification of the original PCR technique is the incorporation of a thermostable DNA polymerase into the PCR protocol which obviates the need for repeated enzyme additions and permits elevated annealing and primer extension temperatures, this enhances the specificity of the primer/template association. Several thermostable DNA polymerases have also been discovered and commercialized, such as the thermostable DNA polymerase from
Pyrococcus furiosus
(Pfu DNA polymerase; U.S. Pat. No. 5,545,552, hereby incorporated by reference), the thermostable DNA polymerase from Thermus flavus (Tfl DNA polymerase; Epicentre Technologies), the thermostable DNA polymerase from
Thermus thermophilus
(Tth DNA polymerase, Epicentre Technologies, Madison, Wis.), a mixture of Taq DNA polymerase and Pyrococcus species GB-D thermostable DNA polymerase (ELONGASE™, Life Technologies, Inc., Gaithersburg, Md.), the thermostable DNA polymerase from
Thermococcus litoralis
(Vent
R
® DNA polymerase, New England Biolabs, Beverly, Mass.), and AMPLITHERM™ DNA polymerase (proprietary thermostable DNA polymerase, Epicentre Technologies). Thermostable DNA polymerases thus serve to increase the specificity and simplicity of PCR.
Another type of cloning method is the so-called “ligation independent cloning” in which a PCR product is produced, purified, denatured along with a recipient DNA, which is then subsequently hybridized to the recipient DNA and results in a nicked or linear recombinant DNA molecule. Because the resulting recombinant molecules are not in a closed circular form they have to be introduced into the proper host cell in order to allow the repair mechanism of the host cell to produce the closed circular recombinant DNA molecule to allow for it to be amplified. Therefore, the cloning efficincy of this method is several orders of magnitude lower than other conventional cloning methods.
As discussed above conventional cloning methods are relatively complex procedures that suffer from drawbacks that make them not amenable to automation. They are labor-intensive and time-consuming. Therefore, a method for automated molecular cloning and amplification of a closed circular recombinant DNA in a single process is desired and provides for a significant advancement in the art.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a method of in vitro automated molecular cloning of a selected donor DNA that has been inserted into a closed circular recipient DNA.
The invention also provides a method of in vitro automated molecular cloning of a selected segment DNA that has been deleted from a closed circular recipient DNA.
The invention further provides a method of in vitro automated molecular cloning of a selected donor DNA that substitutes for a selected segment of a closed circular recipient DNA.
The method for the in vitro automated molecular cloning and amplification of a closed circular nucleic acid comprising:
(a) mixing an effective amount of a donor nucleic acid, a recipient nucleic acid, a pair of 5′-phosphorylated cloning primers, a thermostable DNA ligase, a thermostable DNA polymerase, all four deoxyribonucleoside triphosphates, and an appropriate buffer comprising any cofactor required for activities of both the ligase and polymerase, to result in a cloning mixture; and
(b) thermocycling said cloning mixture through a selected number of cycles at:
(i) a temperature suitable for denaturing the nucleic acids,
(ii) a temperature suitable for annealing the cloning primers to the denatured donor nucleic acid,
(iii) a temperature suitable for polymerase-catalyzed extension of the cloning primers,
(iv) repeating (i) to (iii) to form a pair of insertion primers containing the donor nucleic acid to be inserted,
(v) a temperature suitable for denaturing the nucleic acids,
(vi) a temperature suitable for annealing the insertion primers to the denatured recipient nucleic acid, and
(vii) a temperature suitable for ligase-catalyzed closing of the extended insertion primers, which results in an amplified closed circular nucleic acid recombinant clone containing the donor nucleic acid.
In one illustrative embodiment of the invention, the temperature suitable for polymerase-catalyzed extension of the primers is the same as the temperature suitable for ligase-catalyzed closing of the extended primers. The temperature suitable for annealing the primers to the denatured template can also be the same as the temperature suitable for polymerase-catalyzed extension of the primers.
In preferred embodiments, the cloning mixture is held at the temperature suitable for denaturing the template for about 1 second to 2 minutes in each cycle; the cloning mixture is held at the temperature suitable for annealing the primers to the denatured template for about 1 second to 5 minutes in each cycle; and the cloning mixture is held at the temperature suitable for polymerase-catalyzed extension of the primers and ligase-catalyzed closing of the extended primers for about 1 to 20 minutes in each cycle.
By designing particular cloning primers, the present invention can be used to obtain amplified closed circular nucleic acid clones with inverted or non-inverted insertions or substitutions with a donor nucleic acid fragment, or a deletion of a fragment of the closed circular nucleic acid. When the 3′ and 5′ portions of the pair of cloning primers are complementary to different strands(+/− or −/+) of the donor and recipient DNA, the resulting amplified closed circular clone comprises a donor sequence inserted in an inverted orientation. When the 3′ and 5′ portions of the pair of cloning primers are complementary to the same strands(+/+ or −/−) of the donor and recipient DNA, the resulting a
Chen Zhidong
Ruffner Duane E.
Fredman Jeffrey
Salus Therapeutics, Inc.
Thorpe North & Western, LLP.
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