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  Method for generating transcriptionally active dna fragmentsUSPTO Application #: 20060240469Title: Method for generating transcriptionally active dna fragments Abstract: A method for producing transcriptionally active DNA molecules, comprising (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 5′ 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 3′ end of F2 and a second region complementary to the 3′ end of said DNA molecule. (end of abstract) Agent: Knobbe Martens Olson & Bear LLP - Irvine, CA, US Inventors: Xiaowu Liang, Philip L. Felgner USPTO Applicaton #: 20060240469 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20060240469. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The present application is a continuation of U.S. patent application Ser. No. 09/919,758, filed Jul. 31, 2001, which is a continuation of U.S. patent application Ser. No. 09/535,262, filed Mar. 23, 2000, now U.S. Pat. No. 6,680,977, which claims priority to U.S. Provisional Application Ser. No. 60/125,953, filed Mar. 24, 1999, each of which is hereby expressly incorporated by reference in its entirety. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] 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. [0004] 2. Description of the Related Art [0005] 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. [0006] 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. [0007] The present invention provides a simple, rapid method for the generation of transcriptionally active DNA fragments. SUMMARY OF THE INVENTION [0008] 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. BRIEF DESCRIPTION OF THE DRAWINGS [0009] FIG. 1 is a schematic diagram of the gene amplification method of the present invention. Oligonucleotide primers P1 and P2 are complementary to the 5' end of a promoter sequence (F1) and the 3' end of a terminator sequence (F2), respectively. P3 and P4 are oligonucleotide primers which contain regions complementary to opposite ends of Gene X. In addition, P3 and P4 contain regions which are complementary to the 3' region of F1 and the 5' region of F2. An excess amount of primers P1 and P2 are combined with Gene X, or with a mixture of DNA containing Gene X, and with F1, F2, P3 and P4, or with a mixture of DNA containing F1, F2 and Gene X which has been PCR amplified using P3 and P4, and subjected to PCR to produce a transcriptionally active linear DNA fragment containing F1, F2 and gene X. [0010] FIG. 2 is a schematic diagram showing DNA fragment F1 which contains a thymidine base immediately preceding the region of complementarity with the PCR intermediate (primer P3). [0011] FIG. 3 is a schematic diagram showing the use of peptide nucleic acid (PNA) sequences and PNA clamps to protect the ends of linear expression DNA fragments. Primers P1 and P2 contain a PNA tail which is resistant to proteolytic and exonuclease degradation. The resulting linear expression DNA fragment contains a PNA tail at each of the 5'-ends. The 3' ends are protected by addition of a PNA "clamp" which is also resistant to proteolytic and exonuclease degradation. [0012] FIG. 4 is a schematic diagram showing the use of a PNA "clamp" tail to protect the 5' ends of the linear expression DNA fragment, followed by formation of a clamp during PCR to protect the 3' ends. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0013] The present invention provides a simple, efficient method for generating transcriptionally active DNA fragments which can be readily transfected into animal cells or tissues by conventional nucleic acid transfection techniques, without the need for subcloning into expression vectors and purification of plasmid DNA from bacteria. The transcriptionally active DNA fragments are synthesized by polymerase chain reaction (PCR) amplification of any gene of interest using nested oligonucleotide primers and two DNA fragments, one of which comprises an active transcription promoter sequence and one of which comprises a basic transcription terminator element. A first primer is complementary to the DNA fragment comprising the promoter; a second primer is complementary to the DNA fragment comprising the terminator; a third primer is complementary to both the promoter sequence and one end of the gene of interest; and a fourth primer is complementary to both the terminator sequence and the other end of the gene of interest. These promoters, genes and terminators are linked in an expression cassette as shown in FIG. 1. In addition, the ends of linear DNA containing the expression cassette can be protected from exonuclease digestion during and after transfection by incorporating peptide nucleic acid (PNA) sequences and PNA clamps as shown in FIGS. 3 and 4. [0014] As used herein, the term "promoter" is a DNA sequence which extends upstream from the transcription initiation site and is involved in binding of RNA polymerase. The promoter may contain several short (<10 base pair) sequence elements that bind transcription factors, generally dispersed over >200 base pairs. A promoter that contains only elements recognized by general and upstream factors is usually transcribed in any cell type. Such promoters may be responsible for expression of cellular genes that are constitutively expressed (sometimes called housekeeping genes). There are also tissue-specific promoters limited to particular cell types, such as the human metallothionein (MT) promoter which is upregulated by heavy metal ions and glucocorticoids. [0015] As used herein, the term "terminator" is a DNA sequence represented at the end of the transcript that causes RNA polymerase to terminate transcription. This occurs at a discrete site downstream of the mature 3' end which is generated by cleavage and polyadenylation. [0016] The present method can be used to quickly generate nuclease-resistant and transcriptionally active linear DNA molecules which express any desired gene with any known sequence. The linear DNA can then be delivered into animal cells or tissues for functional analysis, vaccination and other pharmaceutical applications. This method also avoids problems associated with bacterial growth such as toxicity and stability. This method can also be completely automated for use in high-throughput screening methods. [0017] Referring now to FIG. 1, oligonucleotide primers P1 and P2 are complementary to the 5' region of DNA fragments F1 and F2, respectively. Fragment F1 comprises a transcription promoter (darkened region) and fragment F2 comprises a transcription terminator (darkened region). Primers P3 and P4 are complementary to the 3' ends of F1 and F2, respectively, and to different ends of the DNA fragment containing Gene X. FIG. 1 shows the putative intermediates and the final product which are formed in the present PCR-based method when P1, P2, P3, P4 , F1 and F2 are added to a DNA template comprising Gene X. It should be noted that amount of primers P1 and P2 present in the reaction mixture are preferably at least about 100 fold greater than the amounts of P3, P4, F1 and F2, to minimized the amounts of intermediate products generated during the reaction. To generate the first intermediate, primer P3 and P4 are used which amplify from the promoter and terminator sequences of F1 and F2 across Gene X, resulting in an intermediate having the promoter and terminator sequences on the ends and Gene X in between these sequences. [0018] The second intermediate is formed by hybridization of the first intermediate with fragments F1 and F2 via their complementary promoter and terminator sequences (darkened regions), followed by PCR amplification from primers P3 and P4, resulting in a DNA segment comprising the entire F1, F2 and Gene X. [0019] In the last step of the reaction, PCR amplification of the second intermediate using primers P1 and P2 results in amplified amounts of the complete transcriptionally active DNA fragment. [0020] The method of the invention may be performed by adding all of the components in a single reaction mixture. Alternatively, two separate PCR reactions can be performed. In the first reaction, the template gene, P3 and P4 are used first. The product of this reaction is then used as a template for a second PCR reaction involving fragments F1 and F2, plus primers P1 and P2. Continue reading... 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