Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-03-23
2001-08-28
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S320100, C435S091100, C514S04400A
Reexamination Certificate
active
06280977
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for generating transcriptionally active DNA fragments. More specifically, the method relates to synthesis of a DNA fragment by polymerase chain reaction (PCR) using nested primers, promoter sequences and terminator sequences.
2. Description of the Related Art
In addition to the tremendous progress made in the past few years in sequencing the human genome, efforts have also been made to sequence other organisms that are of biomedical importance. For example, complete genomic sequences have been obtained for
Borrelia burgdorferi
(cause of Lyme disease), Chlamydia,
Heliobacter pylori
and
Mycobacterium tuberculosis
. The fast-growing sequence information provides immense opportunities to reveal the basic biology of related organisms at the gene/molecular level and to develop novel therapeutics or vaccines against various pathogens.
However, this vast sequence information also mandates a much more efficient and streamlined way to screen and identify genes of interest from tens of thousands of candidate genes. The conventional approach to gene screening and identification involves generation of a cDNA library, subcloning the DNA inserts into plasmid vectors (expression vectors), purifying plasmid DNA from bacteria for each individual cDNA clone and transfecting animal cells or tissues for functional analysis of the encoded gene product. This method, even in conjunction with the use of polymerase chain reaction (PCR) to generate cDNA fragments to allow directional and in-frame cloning, is still time consuming, costly and difficult to automate.
The present invention provides a simple, rapid method for the generation of transcriptionally active DNA fragments.
SUMMARY OF THE INVENTION
One embodiment of the present invention is a method for generating a transcriptionally active DNA molecule, comprising polymerase chain reaction (PCR) amplification of said DNA molecule in the presence of a first DNA fragment (F1), second DNA fragment (F2), first primer (P1), a second primer (P2), a third primer (P3) and a fourth primer (P4) wherein: F1 comprises a promoter sequence; F2 comprises a terminator sequence; P1 is complementary to the 5′ end of F1; P2 is complementary to the 3′ end of F2; P3 comprises a first region complementary to the 3′ end of F1 and a second region complementary to the 5′ end of said DNA molecule; P4 comprises a first region complementary to the 5′ end of F2 and a second region complementary to the 3′ end of said DNA molecule, whereby a transcriptionally active DNA molecule is produced by said PCR amplification. Preferably, F1 is the cytomegalovirus IE promoter. In one aspect of this preferred embodiment, the transcriptionally active DNA molecule encodes a therapeutic gene. The method may further comprise the step of adding a PNA tail to the 5′-end of P1 and P2 prior to the PCR amplification. Preferably, a thymidine base immediately precedes the region of complementarity between the third primer P3 and the first DNA fragment F1. In another aspect of this preferred embodiment, a thymidine base immediately precedes the region of complementarity between the fourth primer P3 and the second DNA fragment F2. The method may also further comprise the step of adding a PNA clamp to said transcriptionally active DNA molecule after said PCR amplification. Preferably, the method further comprises the step of adding a PNA molecule via a linker (PNA clamp tail) to primers P1 and P2 prior to the PCR amplification.
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Felgner Philip L.
Liang Xiaowu
Einsmann Juliet
Gene Therapy Systems
Jones W. Gary
Knobbe Martens Olson & Bear LLP
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