BAC-CLONING AND MUTAGENESIS OF HERPESVIRUS GENOMES
Cloning as an infectious plasmid and mutagenesis in E.coli has become the method of choice for genetic analysis of viruses with small genomes. Until recently, this approch was not accessible for herpesviruses since their large genomes (150 to 230 kbp) could not be cloned in one piece in E.coli....
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http://www.lmb.uni-muenchen.de/groups/messerle/bac.htmOnly genome sequences determined by systematic bacterial artificial chromosome (BAC) clone sequencing will have clones available. Whole Genome Shotgun (WGS) clones are generally not available for ordering, but if the genome sequences are the result of a composite assembly, BAC clones may be available in certain regions. The human genome sequence however has been solely determined by systematic BAC clone sequencing...
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How to identify an address:
The membrane is divided into 6 fields (diagram provided). Each field contains 384 squares. The 384 squares represent the row and column identification of the BAC. Within each square there are 16 positions where 8 clones are spotted in duplicate (diagram). The pattern of the spotted clones will generate the plate address of the BAC. To identify your clone, please follow the directions below.
The most complicated part about identifying a clone address is that consecutive plates are not spotted into each field. The 384 well plates are spotted onto the membrane with plates 1-6 spotted into fields 1-6 respectively (duplication pattern 1, see diagram). Since there is a total of 6 fields on the membrane, the cycle will continue with the next six consecutive plates (plates 7 through 12) again being spotted into fields 1 through 6 respectively, but in a different duplication pattern (duplication p.....
The purpose of this manual is to explain what the filters are, clone designation procedure, what screening conditions to use, and clone analysis for the Clemson University Arabidopsis BAC library. Filter users are urged to send clone information to schoi@clemson.edu for deposition into the BAC web depository.
The construction of bacterial artificial chromosome (BAC) libraries
SANGDUN CHOI 1,2 and ROD A. WING 2
1 Department of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843, USA
2 Departments of Agronomy & Biological Sciences, Clemson University Genome Center, Clemson University, Clemson, SC 29634, USA
Introduction
Cloning of exogenous DNA into bacterial artificial chromosomes (BACs) provides a new approach to the analysis of the genomes of higher organisms [1]. BAC libraries containing large genomic DNA inserts are important tools for positional cloning, physical mapping and genome sequencing. A number of human and plant BAC libraries have been constructed (e.g., human: [2], Arabidopsis: [3], rice: [4], sorghum: [5]). Bacterial artificial chromosome vectors utilize the E. coli single-copy fertility plasmid and can maintain genomic DNA fragments up to 350 kb. Very little or no rearrangement of the inserts or chimerism have been observed [1, 5, 6, 7]. Other systems for the cloning of large DNA fragments have been developed. The development of yeast artificial chromosome vectors (YAC: [8]) permits cloning of fragme1. BACs are usually supplied as stabs in agar (2.5 ml screw-top tubes filled with LB agar, stabbed with a toothpick which has been put into a bacterial colony). These are then frozen; at -70C they will keep indefinitely, at -20C for a year or more, at 4C for several months and at room temperature (e.g. during shipping) for a week.
2. Make up 10 ml LB broth + 3 ul of chloramphenicol (dissolved in alcohol, 50 mg/ml, so final concentration is 15 ug/ml, stored at 4C) for each stab which will be grown up....
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http://www.le.ac.uk/biology/phh4/bacclone.htm
| 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.
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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..
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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.
| File Format: Microsoft Word - View as HTML Among these are techniques like primed in situ (PRINS) labeling or FISH with ... The probes are then PCR-labeled through the incorporation of either ... www.aecom.yu.edu/home/hgp/GIF%20techniques.doc |
| File Format: Microsoft Word - View as HTML In situ hybridization 1: Tissue Fixation, dehydration and embedding, .... Purify the PCR reaction using RNA only silica spin columns (i.e. not the kits that ... www.csulb.edu/~smalcomb/documents/Simon_in_situ.doc |
| File Format: Microsoft Word - View as HTML In situ hybridization. Brains were removed immediately after decapitation, ... Each predicted RT-PCR product spanned an intron/exon junction. ... www.nature.com/nature/journal/ |
| File Format: Microsoft Word - View as HTML Supplemental Protocol for RNA in situ hybridization in fly larval/pupal/adult ... Probes are PCR amplified and cloned into the Topo vector for verification. ... www.biomedcentral.com/content/ |
| File Format: Microsoft Word - View as HTML We recommend the use of an unequivocal nomenclature to refer to in situ hybridization (Pv), RT-PCR (PvRT) and immunocytochemical (PV) data. ... www.columbia.edu/.../faculty/yuste/petilla/ |
| in situ PCR. protocol. and. is detected. by a nonra-. dioactive. immunohistochemical ..... by in situ hybridiza-. tion. after PCR-ampliflcation. Am J Pathol ... www.clinchem.org/cgi/reprint/40/3/381.pdf - |
| File Format: PDF/Adobe Acrobat - View as HTML DIRECT In situ PCR. Labeled PCR. Reaction Mix. Tissue attached to. microscope slides ... In situ PCR: Examples. Human papilloma virus type 16 in ... www.surrey.ac.uk/SBMS/ACADEMICS_homepage/ |
| In situ cDNA synthesis and PCR were conducted by pricking pathogen-invaded epidermal cells (HES) or appressoria (FIS) of the pow- ... www.springerlink.com/index/LYWP8031LWNE7TM9.pdf - |
| rect in situ PCR techniques utilizing a single pair of. primers, most probably due to the ... situ PCR reaction must be monitored by several reliable ... www.springerlink.com/index/M534142784438042.pdf |
| groups have published protocols and data using in situ PCR techniques. (5). This article will discuss some of the basic concepts of in situ PCR, the proto- ... www.genome.org/cgi/reprint/4/4/S151.pdf - |
| File Format: PDF/Adobe Acrobat - View as HTML agriculture, not adequately covered in most textbooks available. Emili Montesinos. University of Girona. In Situ PCR Techniques ... www.im.microbios.org/05march99/14%20Book%20reviews.PDF |
| File Format: PDF/Adobe Acrobat - View as HTML The variables for in situ hybridization and for PCR were ... Key words Hyphomycetes · In situ hybridization · In situ PCR · Rock inhabiting ... www.im.microbios.org/03setember98/08%20Sterflinger.pdf - |
IN SITU PCR AMPLIFICATION SYSTEM -
LA HAFF, JG ATWOOD - 1993 - patents1.ic.gc.ca
... ABSTRACT: Abstract of the Disclosure A complete in situ PCR system for amplification
of nucleic acids contained in a prepared cell or tissue sample. ...
Cited by 35
| To overcome this problem, we designed an in situ PCR technique that links PCR ... first report of in situ PCR on phytopathogenic fungal material. ... journals.cambridge.org/production/ |
| Method: in situ PCR expression analysis (method details to be added). There have been 10374 hits since 09/26/2003. Please note: suggested browsers are ... www.floralgenome.org/fgp/methods/method_in_situ.html - 10k - |
2nd ed, by Gerald Nuovo, 449 pp, with illus, $80, New York, NY, Raven Press, 1994.
This book represents a renewed effort to bring procedures in molecular biology into the domain of anatomic pathology research. For pathologists who cut their teeth on avidin-biotin immunohistochemistry and only recently became comfortable with polymerase chain reaction (PCR) techniques, this book is a logical entry point into leading edge molecular diagnostics and research using PCR in situ hybridization...
http://findarticles.com/p/articles/mi_qa3725/is_199707/ai_n8764476
Authors: Alzahrani A.J.; Vallely P.J.; McMahon R.F.T.1
Source: Journal of Virological Methods, Volume 99, Number 1, January 2002 , pp. 53-61(9)
Publisher: Elsevier
http://www.ingentaconnect.com/content/els/01660934/2002/00000099/00000001/art00383
REVIEW |
Correspondence to: Gerard J. Nuovo, Dept. of Pathology, Ohio State U. Medical Center, S305 Rhodes Hall, 450 W 10th Ave., Columbus, OH 43210. E-mail: gnuovomd@pol.net
| Summary |
|---|
In situ amplification permits the histological localization of low-copy DNA and RNA targets. However, in many instances it would be useful to know the specific phenotype of the target-containing cell or to ascertain the distribution of a different nucleic acid sequence in the same tissue section. This review describes a methodology that allows co-in situ localization of two nucleic acid targets or a DNA/RNA sequence and a protein in paraffin-embedded, formalin-fixed tissue. The key variable for detection of low-copy RNA targets by RT in situ PCR is optimal protease digestion to permit cDNA target-specific incorporation of the reporter nucleotide. This is achieved via inactivation of nonspecific DNA synthesis by overnight DNase digestion. The key variable for immunohistochemical localization of proteins is to determine the effect of protease digestion on the antigen-based signal intensity. Background for DNA targets by in situ hybridization or, for targets present in 1–10 copies per cell, PCR ISH is dependent primarily on probe concentration and the stringency of the post-hybridization wash. Radioactive 3H-labeled nucleotides permit an excellent distinction with colorimetric signals for co-localization, although two distinct chromogens can in many instances allow successful localization of two different targets.
1 Jonathan A Terrett,1 and Paul J Adam1 Corresponding author.
