Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Utility Patent
1996-01-31
2001-01-02
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S912000, C435S173300, C435S242000, C536S023100
Utility Patent
active
06168918
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of detecting the presence of stably integrated plasmid DNA sequences in eukaryotic chromosomal DNA in samples that contain a mixture of integrated and free (non-integrated) plasmid DNA molecules.
BACKGROUND OF THE INVENTION
The fields of gene therapy and genetic vaccination include protocols in which nucleic acid molecules, including plasmid DNA molecules and viral genomes, are used as an active agent. The nucleic acid molecules encode proteins that have therapeutic and/or prophylactic effects on the individuals to whom the nucleic acid molecules are delivered. For example, PCT/US94/00899, which is incorporated herein by reference, and the U.S. parent applications to which it claims priority, which are also each incorporated herein by reference, disclose genetic immunization protocols using plasmid DNA delivered in conjunction with compounds that facilitate DNA uptake. PCT/US90/01515, which is incorporated herein by reference describes delivery of naked plasmid DNA, i.e. DNA that is free from any agents which facilitate uptake. Others teach the use of liposome mediated DNA transfer, DNA delivery using microprojectiles (U.S. Pat. No. 4,945,050 issued Jul. 31, 1990 to Sanford et al., which is incorporated herein by reference), and DNA delivery using electroporation. In each case, the DNA may be plasmid DNA that is produced in bacteria, isolated and administered to the animal to be treated. The plasmid DNA molecules are taken up by the cells of the animal where the sequences that encode the protein of interest are expressed. The protein thus produced provides a therapeutic or prophylactic effect on the animal. The use of viral vectors and other means of delivering nucleic acid molecules to cells of an individual in order to produce a therapeutic and/or prophylactic immunological effect on the individual are similarly well known.
Gene therapy and genetic immunization involve the transfer of DNA into cells and the nuclear compartment of cells in vivo. Some forms of gene therapy and DNA vaccination rely on well known methods to insert (integrate) the plasmid DNA into a eukaryotic chromosome. This is carried out to enhance the amount and longevity of DNA expression. Alternatively, DNA might be transferred in a form which does not intentionally integrate, but instead express as an episomal element. This is preferred in genetic immunization procedures, due to the possible consequence or an uncontrolled integration event.
In particular, there is a risk that the transferred DNA will integrate into the chromosome of the eukaryotic cell at a site in the chromosome which results in the cell displaying an abnormal phenotype. Translocations and integration of foreign DNA have been observed in many transformed cells. The observation that integration of foreign DNA into chromosomal DNA can lead to a transformed phenotype is a major concern in protocols which use foreign DNA as an active agent to be delivered to cells. In protocols in which plasmid DNA is used as an active agent, the possibility of plasmid DNA integration into chromosomal DNA presents a potential risk which must be assessed.
There is a need for a highly sensitive and specific assay which can reliably detect the presence of stably integrated DNA sequences in eukaryotic chromosomal DNA in samples which also contain the same DNA sequences in an episomal form which is non-integrated. There is a need for a highly sensitive and specific assay which can reliably detect the presence of stably integrated plasmid DNA sequences in eukaryotic chromosomal DNA in samples which also contain the same plasmid DNA sequences in a form which is non-integrated. There is a need for a highly sensitive and specific assay which can reliably detect the presence of stably integrated viral DNA sequences in eukaryotic chromosomal DNA in samples which also contain the same viral DNA sequences in an episomal form which is non-integrated.
SUMMARY OF THE INVENTION
The present invention provides a method to test whether or not foreign DNA, i.e. plasmid DNA or viral genomic nucleic acid molecules, that is introduced into eukaryotic cells integrates into the chromosomal DNA of the eukaryotic cells. The method provides a simple and efficient means for the identification of integrated foreign DNA sequences in samples which also contain free (non-integrated) foreign DNA molecules. The present invention relates to methods of detecting the presence of foreign DNA that is integrated into eukaryotic chromosomal DNA in a sample that contains free foreign DNA molecules and chromosomal DNA molecules from eukaryotic cells.
According to the invention, the foreign DNA must have certain characteristics. The foreign DNA which can be detected in the method of the invention has at least one DpnI restriction enzyme site as well as one or more non-DpnI restriction enzyme sites which can be cut by non-DpnI restriction enzymes. The foreign DNA molecules must be produced in a cell that has the enzyme deoxyadenosine methyltransferase (dam) so that the foreign DNA has the methylation pattern that is associated with DNA produced in dam
+
cells.
According to the invention, the eukaryotic cells into which the free foreign DNA molecules are introduced must not have the enzyme dam so that chromosomal DNA does not have the methylation pattern that is associated with DNA produced in dam
+
cells.
The method of the invention comprises a series of steps. The total DNA from eukaryotic cells that contain plasmid DNA is digested with one or more of the non-DpnI restriction enzymes that can cleave the foreign DNA to produce DNA digestion segments of multiple sizes. The DNA digestion segments are then fractionated to produce a plurality of fractions of DNA digestion segments. Fractionation results in the separation of foreign DNA sequences which were integrated from the bulk of the remainder of the chromosomal DNA. In addition, free foreign DNA sequences are also fractionated in a similar manner. The DNA digestion segments in each fraction are then digested with restriction enzyme DpnI. Following DpnI digestion, fragments of foreign DNA are amplified using sets of primers that flank a DpnI restriction enzyme site in the foreign DNA. Free, non-integrated foreign and native chromosomal DNA sequences are not amplified. Detection of the presence of amplified fragments indicates that foreign DNA has integrated into eukaryotic chromosomal DNA.
Some preferred embodiments of the present invention provides a method to test whether or not plasmid DNA that is introduced into eukaryotic cells integrates into the chromosomal DNA of the eukaryotic cells. The method provides a simple and efficient means for the identification of integrated plasmid DNA sequences in samples which also contain free (non-integrated) plasmid DNA molecules. Some preferred embodiments of the present invention relates to methods of detecting the presence of plasmid DNA that is integrated into eukaryotic chromosomal DNA in a sample that contains free plasmid DNA molecules and chromosomal DNA molecules from eukaryotic cells.
According to the invention, plasmid DNA must have certain characteristics. The plasmid DNA which can be detected in the method of the invention has at least one DpnI restriction enzyme site as well as one or more non-DpnI restriction enzyme sites which can be cut by non-DpnI restriction enzymes. The plasmid DNA molecules must be produced in a cell that has the enzyme deoxyadenosine methyltransferase (dam) so that the plasmid DNA has the methylation pattern that is associated with DNA produced in dam
+
cells.
According to some of the preferred embodiments of the invention, the eukaryotic cells into which the free plasmid DNA molecules are introduced must not have the enzyme dam so that chromosomal DNA does not have the methylation pattern that is associated with DNA produced in dam
+
cells.
Some of the preferred embodiments of the method of the invention comprises a series of steps. The total DNA from eukaryotic cells that contain plas
Ciccarelli Richard Benjamin
Pachuk Catherine Julia
Satishchandran C.
American Home Products Corp.
Fredman Jeffrey
Howson and Howson
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