| File Format: PDF/Adobe Acrobat - View as HTML ates the linear expression template via overlap extension PCR, .... Figure 14: Scheme of domain fusion by overlap extension PCR using the RTS E. coli Linear ... www.roche-applied-science.com/sis/proteinexpression/literature/manual/chapter1/RTS_30_35.pdf |
| In conclusion, a modified overlap extension PCR technique. is described to improve the construction of multiple. chimeric genes and proteins, site-directed ... www.springerlink.com/index/LWN2032286220T71.pdf - |
Overlap extension PCR
RIKEN Yokohama Institute, Yokohama, Japan.
Sequencing of the human genome in combination with computational annotation has provided tremendous data on predicted genes. However, for most of them, no corresponding cDNAs are available yet. Furthermore, even where cDNA clones were obtained, gene transcripts often have many different splice variants that are not covered by current gene collections. For direct synthesis of cDNA clones corresponding to predicted genes, new splice variants, or any other gene of interest, we established optimal PCR conditions for the direct amplification of exons from genomic DNA, which require a specific Taq aptamer. PCR products comprising differently tagged exons were concatenated by overlap extension into full-length cDNAs. To prove the effectiveness of the approach, the 1900-bp full-length open reading frame of the human mitochondrial aldehyde dehydrogenase (ALDH2) gene was synthesized in a two-step reaction comprising all 13 exons. Thus, our conditions are of general value for in vitro synthesis of cDNAs and alternative splice variants from genomic DNA.
PMID: 15283210 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&list_uids=15283210&cmd=Retrieve&indexed=google
FARRELL DJ, MCKEON M, DAGGARD G, MUKKUR TK.
Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 1999 Sep 26-29; 39: 225 (abstract no. 1569).Queensland Health Pathology Service, Toowoomba, AUSTRALIA
BACKGROUND: Although many PCR methods have been reported, none has yet fulfilled the consensus recommendations for the use of PCR in the diagnosis of B. pertussis (BP) infections (Meade & Bollen, J Med Microbiol 1994; 41:51) and hence a standardised method has not been forthcoming. The aim of this study was to develop such a method.METHODS: A rapid cycle PCR method with a microwell format/probe hybridisation detection step was developed using novel oligonucleotides targeted at the pertussis toxin operon. An internal control was developed using overlap extension PCR and mouse beta-actin DNA. A novel hybridisation stringency ratio (HSR) was developed to differentiate BP from B. parapertussis (BPP) and B. bronchiseptica (BB). Contamination was minimised by the use of heat labile uracil N-glycosylase. Specimens were treated by simply diluting 1:10 in H[2]O and then heating for 20 minutes at 99.9[o]C. Analytical sensitivity and specificity were ascertained using 35 strains of Bordetella species and 30 strains of common respiratory pathogens and commensal organisms. Clinical sensitivity and specificity were ascertained using 79 specimens tested using a nested PCR method targeting the insertion sequence IS481 (Farrell et al., J Clin Microbiol 1999; 37:606).RESULTS: Analytical specificity was 100%. Analytical sensitivity was comparable to nested IS481 PCR (1 organism per reaction). 35/36 nested PCR positive specimens were positive - inhibitory substances being detected in the remaining specimen (which became positive after DNA purification). 2 specimens that fulfilled a clinical definition of pertussis were positive by the new method/negative by IS481 PCR. 41 specimens were negative by both methods. All Bordetella species and clinical specimens were identified correctly to the species level using HSR.CONCLUSION: The following were successfully developed: 1) A PCR method which fulfils all recommendations with the added benefit of being rapid (4 hours) and reproducible. 2) A novel, simple mechanism (HSR) to allow differentiation between BP and BPP/BB post-amplification.
Full Details Here
http://gateway.nlm.nih.gov/MeetingAbstracts/102245122.html
Dept. of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905.
Gene Splicing by Overlap Extension or "gene SOEing" is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5'-ends of the primers, any pair of polymerase chain reaction products can be made to share a common sequence at one end. Under polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another, forming an overlap. Extension of this overlap by DNA polymerase yields a recombinant molecule. This powerful and technically simple approach offers many advantages over conventional approaches for manipulating gene sequences.
PMID: 2357375 [PubMed - indexed for MEDLINE]
| Gene. 1989 Apr 15;77 (1):51-9 2744487 [Cited: 762] | |
| Site-directed mutagenesis by overlap extension using the polymerase chain reaction. | |
| S N Ho , H D Hunt , R M Horton , J K Pullen , L R Pease | |
| Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends. These fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand. The resulting fusion product is amplified further by PCR. Specific alterations in the nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into the overlapping oligo primers. Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility complex class-I gene have been generated, cloned and analyzed. Screening of mutant clones revealed at least a 98% efficiency of mutagenesis. All clones sequenced contained the desired mutations, and a low frequency of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler and approaches 100% efficiency in the generation of mutant product. | |
| Mesh-terms: Amino Acid Sequence; Animals; DNA, Recombinant; DNA-Directed DNA Polymerase; Exons; Gene Amplification; Genes, MHC Class I;Genes, Synthetic; Genetic Engineering, methods; Mice; Molecular Sequence Data; Mutation; Oligodeoxyribonucleotides, chemical synthesis; Oligodeoxyribonucleotides, genetics; Support, Non-U.S. Gov't; Support, U.S. Gov't, P.H.S.; Taq Polymerase; | |
| [Pubmed] [Scholar] [EndNote] [BibTex] http://lib.bioinfo.pl/pmid:2744487 | |
| Rapid and Efficient Site-directed Mutagenesis by the Single-tube Megaprimer PCR Method | |
| Site-specific Mutagenesis by Overlap Extension | |
| In Vitro Mutagenesis Using Double-stranded DNA Templates: Selection of Mutants with DpnI | |
| Genetic Engineering with PCR | |
| Mutagenic PCR | |
| Rapid PCR Site-Directed Mutagenesis | |
| PCR-Mediated Gene Disruption: One-Step Method |
| Purification of Genomic DNA from Whole Blood: A Solution-Based Method | |
| A Silica Membrane-Based Method for the Isolation of Genomic DNA from Tissues and Cultured Cells | |
| DNA IQ Isolation of Genomic DNA from Stains and Buccal Swabs |
1) Add the following to a microfuge tube:
In this experiment, polymerase chain reaction (PCR) is used to amplify a nucleotide sequence from chromosome 8 to look for an insertion of a short DNA sequence called Alu within the tissue plasminogen activator (TPA) gene. Although the DNA from different individuals is more alike than different, there are many regions of the human chromosomes t..
Edited by:
http://www.mcb.uct.ac.za//pcrconcn.htm
PCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules. The sensitivity of this technique means that the sample should not be contaminated with any other DNA or previously amplified products (amplicons) that may reside in the laboratory environment.
Full Protocol Here
Materials:Program a final extension at 72 degrees C for 7 min.
sterile water 10X amplification buffer with 15mM MgCl2 10 mM dNTP 50 μM oligonucleotide primer 1 50 μM oligonucleotide primer 2 5 unit/μl Taq Polymerase template DNA (1 μg genomic DNA, 0.1-1 ng plasmid DNA) in 10 μl mineral oil (for thermocyclers without a heated lid 1. Combine the following for each reaction (on ice) in a 0.2 or 0.5 ml tube:
10X PCR buffer
10 μl
Primer 1
1 μl
Primer 2
1 μl
dNTP
2 μl
template DNA and water
85.5 μl
Taq Polymerase
0.5 μl
2. Prepare a control reaction with no template DNA and an additional 10 μl of sterile water.
3. If the thermocycler does not have a heated lid, add 70-100 μl mineral oil (or 2 drops of silicone oil) to each reaction.
4. Place tubes in a thermal cycler preheated to 94 degrees C.
5. Run the following program:
for 30 cycles.
