Gene Cloning and Analysis Chapter Abstracts
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Chapter 1. ADJUSTMENT OF PARAMETERS IN THE YEAST TWO-HYBRID SYSTEM: CRITERIA FOR DETECTING PHYSIOLOGICALLY SIGNIFICANT PROTEIN-PROTEIN INTERACTIONS.
Erica A. Golemis, Ilya Serebriiskii and Susan F. Law
In vivo transcription-based assays for protein-protein interactions such as the Two-Hybrid System are powerful methods for identifying novel proteins based on their physical association with known proteins of biological interest, or for characterizing the degree and nature of interactions between sets of proteins. Because of the complexity inherent in assays taking place within a living organism, a key issue for the effective use of two-hybrid approaches is the ability to determine whether apparent interactions are likely to be physiologically relevant.
In this chapter, a number of the different two-hybrid systems currently available for use will be reviewed. Then, taking as a model one such system, the Interaction Trap, examples of different reagents for use in varying the affinity range of detectable interactions will be outlined. Also set forth are a number of protocols to establish an appropriate set of conditions for either screening a library or analysing the interaction phenotype between protein sets. Finally, a number of general guidelines are suggested for trouble-shooting two-hybrid results, and for eliminating falsely positive interactions.
Chapter 2. pJUFO: A PHAGE SURFACE DISPLAY SYSTEM FOR CLONING GENES BASED ON PROTEIN-LIGAND INTERACTION
Reto Crameri
The phage surface display technology, which provides a link between genetic information and gene product, is becoming an important tool in biotechnology. Cloning systems designed to express gene products on the surface of phage have proven to be useful for the display and screening of small peptides, as well as functional monomeric, homo- or heterodimeric proteins. Unfortunately, the available systems do not allow the construction of cDNA expression libraries, because translational stop codons present at the 3' non-translated regions of eukaryotic mRNA prevent generation of fusion proteins N-terminal to phage coat proteins. Fusion to the membrane-bound C-terminus of the coat protein would hamper incorporation of the fusion product into the virion. To covalently link the expressed cDNA products to the phage surface, a strategy based on the phagemid pComb3 and the ability of the Jun and Fos leucine zippers to heterodimerize was developed. The gIII gene product was modified, allowing the production of a chimaeric protein in which the Jun leucine zipper is fused N-terminal to this viral coat protein. The modified gIII protein was then used to capture cDNA products decorated with the Fos leucine zipper, thus allowing these proteins to become structurally incorporated into phage particles during phage morphogenesis. To avoid interphage exchange of fos-cDNA fusion products, cysteines flanking both leucine zippers were engineered to provide a covalent link between the cDNA gene products and the genetic information required for their production. The resulting phagemid, pJuFo, allows expression and display of cDNA libraries on the phage surface. Screening can thus be acomplished by employing the powerful biopanning procedure, which has been very successfully used with other filamentous phage systems.
Chapter 3. RESTRICTION ENDONUCLEASE-BASED GENE EXPRESSION FINGERPRINTING
N.B.Ivanova and A.V.Belyavsky
A method for identification of differentially expressed genes, gene expression fingerprinting (GEF), is described. The GEF protocol involves the following steps: i) generation of the representative amplifiable primary set of cDNA restriction fragments by digestion of the cDNA with a frequently cutting restriction endonuclease, solid phase selection of 3' terminal fragments, and adapter ligation; ii) subdivision of the primary set into simpler secondary subsets by PCR with nested primers and/or by sequential restriction digestion of immobilized labeled cDNA fragments; iii) separation of the secondary subsets by gel electrophoresis in order to resolve the individual fragments and thereby compare the abundances of the corresponding mRNAs between two or more different mRNA populations. Recovery of the fragments of interest is achieved by elution from the gel, oligo (dG)-tailing and re-amplification. With further increases in the resolving power of this approach, it may become possible to reliably identify expressed genes on the basis of the mobility of cDNA fragments, thereby making possible the global analysis of gene expression.
Chapter 4. PNA USED FOR SPECIFIC CAPTURE OF NUCLEIC ACIDS
Martin B. Borre, Michael Naesby and Henrik Ørum
Detection of antigens or antibodies in patient samples are widely used methods in diagnostics. However, these methods tend to lack speed and/or sensitivity or they are dependent on the patient's ability to form specific antibodies. In recent years, molecular biology has offered an attractive alternative: the sequence specific detection of nucleic acids. Such techniques can be extremely sensitive, allowing the detection of the presence of a single DNA molecule. Most of these techniques, however, are dependent on enzymes which can be inhibited by compounds in patient samples. Purification of target DNA prior to amplification is therefore of great importance. In this chapter we describe some general properties of peptide nucleic acid (PNA). Based upon these data, we have designed protocols for PNA-based DNA purification techniques and we demonstrate the use of these protocols in molecular diagnostics.
Chapter 5. A PCR-BASED METHOD FOR ISOLATION OF GENOMIC DNA FLANKING A KNOWN DNA SEQUENCE
Catherine A. Boulter and Dipa Natarajan
We describe a simple PCR-based method for the isolation of genomic DNA that lies adjacent to a known DNA sequence. The method is based on the directional cloning of digested genomic DNA into the multiple cloning site of a pUC-based plasmid to generate a limited genomic library. The library is plated onto a number of selective LA plates which are incubated overnight, and recombinant plasmid DNA is then isolated from resistant colonies pooled from each plate. PCR amplification is performed on the pooled recombinant plasmid DNAs using primers specific for the pUC vector and the known genomic sequence. The combination of efficient directional cloning and bacterial transformation gives relative enrichment for the genomic sequence of interest and generates a simple DNA template, enabling easy amplification by PCR.
Chapter 6. AFFINITY SELECTION OF EUKARYOTIC mRNAS AND FULL-LENGTH
cDNAS BY A CAP RETENTION PROCEDURE (CAPTURE)
Jerry Pelletier
The ability to generate complementary DNA (cDNA) libraries is one of the most fundamental procedures of contemporary molecular biology. A major concern of current methods is that the majority of cDNAs present in any given library are incomplete, rendering the characterization of genes an inefficient and time-consuming task. We have developed an affinity selection method to purify mRNAs via non-covalent capture of the 5' cap structure. This protocol can also be used to enrich reverse transcription reaction products for full-length cDNAs. The key features of this method are a bifunctional fusion protein that can be immobilized onto a solid support matrix and which binds 5' cap structures of eukaryotic mRNAs. Following first strand cDNA synthesis, a single strand specific nuclease is used to remove cap structures from incomplete mRNA:cDNA duplexes. Specific enrichment of complete mRNA:cDNA duplexes is then achieved using this novel affinity matrix. This method can be used to enrich for full-length cDNAs or cDNA clones having complete 5' ends and to generate cDNA libraries in which reverse transcribed products of polyadenylated and nonpolyadenylated mRNAs are equally represented.
Chapter 7. RNA LIGASE-MEDIATED RACE: AN EFFECTIVE METHOD FOR THE CLONING OF FULL-LENGTH cDNA ENDS
Brian C. Schaefer
Rapid Amplification of cDNA Ends (RACE) is a polymerase chain reaction (PCR)-based technique which was developed to facilitate the cloning of full length cDNA 5'- and 3'-ends after a partial cDNA sequence has been obtained by other methods. While RACE can yield complete sequences of cDNA ends in only a few days, the RACE procedure frequently results in the exclusive amplification of truncated cDNA ends, undermining efforts to generate full-length clones. Many investigators have suggested modifications to the RACE protocol to improve the effectiveness of the technique. In this chapter, the major variations of the RACE method are briefly reviewed, with particular emphasis on RNA Ligase-Mediated RACE (RLM-RACE) which offers a number of advantages over other RACE techniques. A detailed protocol for RNA Ligase-Mediated RACE is presented.
Chapter 8. COMBINATORIAL SCANNING SITE-DIRECTED MUTAGENESIS
Jean Chatellier and Thierry Vernet
Our understanding of the complex relationships between protein structure and function has been considerably enhanced by the advent of synthetic oligonucleotide-based in vitro mutagenesis procedures. The role of individual residues or groups of residues at pre-determined sites in a protein can be investigated in detail by combinatorial substitutions, insertions or deletions. Such analysis is facilitated by the replacement of targeted residues by a single type of amino acid, the most frequently used being alanine. Unfortunately, combinatorial amino acid scanning using currently available mutagenesis protocols is restricted to subsets of residues. We propose a protocol that alleviates this limitation by combining the "column splitting" oligonucleotide (oligo) synthesis method with standard in vitro site-directed mutagenesis. The method has been previously validated by performing combinatorial alanine scanning mutagenesis on a set of residues located at the interface between the variable domains of an antibody fragment (1). Escherichia coli mutant libraries were easily screened by restriction analysis of PCR amplified fragments, as facilitated by the incorporation of sets of diagnostic restriction sites within the mutagenic oligo. The occurrence of multiple mutations was found to be in good agreement with the statistically predicted distribution. The present chapter broadens the scope of the initial application of this mutagenesis technique.
Chapter 9. A RAPID METHOD FOR CREATING GENE MUTATIONS AND FUSIONS USING PCR-LIGATION-PCR MUTAGENESIS
Stuart Alvaro Ali and Alexander Steinkasserer
Here we describe a novel PCR method for site-specific mutagenesis. Unlike splice overlap extension (SOE) PCR, this technique does not require primers with overlapping complementary sequences, but still maintains sequence specificity at the recombination junction. Like the PCR process itself, the principle is only inherently obvious after it has been explained. Remarkably, this simple technique can be applied universally to generate gene fusions, deletions, insertions and point mutations. The method is practical, cost effective and reproducible, and it therefore offers many advantages over existing techniques and commercially available mutagenesis kits.
Chapter 10. DOUBLE REPLACEMENT GENE TARGETING IN EMBRYONIC STEM CELLS FOR THE INTRODUCTION OF SUBTLE ALTERATIONS INTO ENDOGENOUS MOUSE GENES
David W Melton
Gene targeting can be used in embryonic stem cells to produce strains of genetically modified mice. Studies employing targeted gene inactivation are making a major contribution to the understanding of mammalian gene function and to the modelling of human inherited genetic deficiency diseases in the mouse. We have developed a procedure called double-replacement gene targeting, which uses hypoxanthine phosphoribosyltransferase (HPRT) selectable markers in HPRT-deficient embryonic stem cells for the production of mice with subtle gene alterations, rather than conventional gene inactivations (knockouts). In the first targeting step, the target locus is inactivated by deleting part of the gene and replacing it with an HPRT selectable marker. In the second step, the HPRT marker is itself replaced by a modified version of the original gene segment, which contains the desired subtle gene alteration. From the initial targeting event, this procedure can be used to create a series of mouse strains with different subtle gene alterations.
Chapter 11. UNIVERSAL TA CLONING
Ming-Yi Zhou and Celso E Gomez-Sanchez
TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase. Taq polymerase has non-template dependent activity which preferentially adds a single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most of the molecules PCR amplified by Taq polymerase possess single 3'-A overhangs. The use of a linearized "T-vector" which has single 3'-T overhangs on both ends allows direct, high-efficiency cloning of PCR products, facilitated by complementarity between the PCR product 3'-A overhangs and vector 3'-T overhangs. The TA cloning method can be easily modified so that the same T-vector can be used to clone any double-stranded DNA fragment, including PCR products amplified by any DNA polymerase, as well as all blunt- and sticky-ended DNA species. This technique is especially useful when compatible restriction sites are not available for the subcloning of DNA fragments from one vector to another. Directional cloning is made possible by appropriate hemi-phosphorylation of both the T-vectors and the inserts. With a single T-vector at hand, any DNA fragment can be cloned without compromising the cloning efficiency. The universal TA cloning method is thus both convenient and labor-saving.
Chapter 12. LONG PCR: RAPID RESTRICTION MAPPING AND SELECTIVE
SUPPRESSION BY RESTRICTION DIGESTION
Chengtao Her and Richard M. Weinshilboum
The polymerase chain reaction (PCR) is the most widely used technique for the study of DNA. Applications for the PCR have been extended significantly by the development of "long" PCR, a technique that makes it possible to amplify DNA fragments up to 40 kb in length. This chapter describes two novel applications of the long PCR technique, one which simplifies restriction mapping and another which enhances amplification specificity and yield. The same primers used to perform the long PCR amplification can be used as probes to perform restriction mapping of the DNA fragment amplified. Restriction digestion performed prior to long PCR amplification can be used to selectively suppress the amplification of members of families of closely related DNA sequences, thereby making it possible to selectively amplify one of a group of highly homologous sequences. These two complimentary techniques, both involving use of the long PCR paired with restriction digestion, have potential application in any laboratory in which the PCR is performed.
Chapter 13. HIGH-RESOLUTION SILVER STAINING-BASED PCR-SINGLE STRAND CONFORMATION POLYMORPHISM (SSCP) ANALYSIS
Langxing Pan and Huaizheng Peng
In this chapter, we describe a high-resolution non-radioactive PCR-SSCP method which utilizes a polyacrylamide-agarose composite gel and background-free silver staining. Addition of agarose into the acrylamide solution produces a strong and flexible gel for SSCP analysis, while treatment of the gel with sodium thiosulfate prior to and after exposure to silver salts results in background-free staining. Together, both modifications improve the resolution of silver staining-based PCR-SSCP analysis and increase the sensitivity of mutation detection to a level similar to that of radioactive labeling-based methods.
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