Genetic Engineering with PCR Chapter Abstracts

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The historical development of techniques for altering genetic information on the molecular level can be divided into three generations: classical genetics, molecular cloning, and polymerase chain reaction-based gene manipulation. These approaches build upon and complement one another, and together form a powerful arsenal of methods for assembling designer genes. Whereas the technology now exists to construct essentially any desired gene sequence, limitations in the understanding of the principles relating protein structure with function preclude the de novo design of gene products. Genetic engineering thus depends to a large degree on modification of the structure of known proteins. A thorough understanding of the fundamental principles of gene manipulation is an important step in the application of genetic engineering.

Chapter 2: Mutagenesis By Megaprimer PCR

Mutagenesis by megaprimer PCR is a simple and versatile method requiring three primers and two rounds of PCR. One primer introduces the specific mutation, while the other two primers flank the region of interest, and can be used in the generation of a set of mutants. The first round of PCR is performed using the mutant primer and one of the flanking primers. The double-stranded product is purified and used as a "megaprimer" in the second round along with the primer flanking the region on the other end. No strand separation of the megaprimer is needed. The wild type DNA is used as template in both PCRs, and therefore, low quantities of DNA from a wide variety of sources can be amplified and mutagenized at the same time. The mutant primer can be designed to incorporate essentially all kinds of mutations, e.g., substitution, deletion, or insertion. A chimeric primer can additionally produce gene fusions through the use of two different genes in the two PCRs. Thus, the generalized megaprimer technique is a highly efficient, flexible, and adaptable procedure for in vitro mutagenesis and recombination.

Chapter 3: Effects of dNTP and Divalent Metal Ion Concentrations on Random PCR Mutagenesis

The error rate in a PCR amplification depends on the respective concentrations of the four deoxynucleoside triphosphates (dNTPs). Therefore, an appropriate combination of these concentrations can transform PCR into a straightforward, efficient and controllable method for random mutagenesis of DNA. A wide range of substitution frequencies can be obtained for the AT-GC and GC-AT transitions as well as for the AT-TA transversion. Alternatively, the dNTP concentrations can in theory be adjusted to improve the fidelity of PCR in applications where random mutations are not desired. In this chapter, rules relating overall mutation rates and relative frequencies of the various base pair substitutions to the dNTP concentrations are established and discussed, and the effect of MnCl₂, a known mutagen, is characterized. The mutagenic effects of several other parameters are reviewed.

Chapter 4: Rapid Cycle Amplification For Construction Of Competitive Templates

The time required for DNA engineering techniques can be significantly reduced with rapid temperature cycling. By air heating and cooling of 10 ul samples in capillary tubes, 30 cycles of amplification can be completed in less than 15 min. Rapid cycle amplification can be used to construct internal competitive templates for quantitative PCR. In most cases, the natural amplicon and its primers are available. Insertions, deletions, or point mutations are introduced into the natural amplicon to allow differentiation of natural and control templates. Only 2 overlap primers are required to insert any sequence from an inner fragment into an outer fragment, with optional replacement of any sequence in the outer fragment. Competitive templates that differ in GC content can be used to distinguish PCR products based on melting curves monitored with fluorescence during PCR. Rapid cycle amplification was used to modify a natural template through insertion of another fragment having different GC content. Alternately, a portion of the natural template was replaced by another fragment. In addition to reducing time and reagent requirements, rapid cycling techniques increase amplification specificity. Rapid cycling is particularly advantageous when low volumes, small sample numbers, and multiple sequential amplifications are required.

Chapter 5: DNA Splicing by Directed Ligation (Sdl)

Splicing by directed ligation (SDL) is a method of inphase joining of PCRgenerated DNA fragments that is based on
a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites. Since these enzymes cleave outside of their recognition sites, the resulting sticky end can have any desired sequence, and the site itself can be removed and does not appear in the final spliced DNA product. SDL is based on the addition of class IIS recognition sites onto primers used to amplify DNA sequences. Cleavage of the PCR products results in elimination of the recognition site-containing flanking sequences and leaves the DNA fragments crowned with protruding ends. With careful design of the sticky ends, several segments can be ligated together in a predetermined order in a single reaction. SDL requires fewer rounds of amplification than overlap extension methods, and is particularly useful for creating a series of recombinants that differ in one segment.

Chapter 6: Multimers of Specific DNA Sequences Generated by PCR

In applying DNA technology, it is often important to exploit the interactions of specific short segments of DNA with other DNAs or proteins in order to capture larger DNA molecules by hybridization, to partially determine DNA sequences by annealing of specific probes, or to purify proteins that bind to specific DNA sequences. Each of these objectives may involve the anchoring of a short specific DNA segment to a solid phase support; in most applications of this type, high quantities of the attached DNA are desirable. If instead of simply anchoring the specific DNA segment, a multimer composed of monomers of the particular sequence is attached to the solid support, many more specific segments can be anchored and made available for interaction. Rather than the conventional view of DNA as a polymer of nucleotides, the polymer or multimer envisioned here consists of monomeric units several nucleotides in length. These sequence monomers are joined in a head-to-tail fashion and repeated many times one after another. Because these multimers require less of the solid support to anchor the same quantity of monomeric DNA segments, there may be less background binding to the support matrix. This approach may also facilitate miniaturization.

Chapter 7: The Use of PCR-based Scanning Mutagenesis in Molecular Cell Biology

The PCR-based interchange of protein coding regions by Homolog-Scanning Mutagenesis (HSM) and substitution of amino acid residues by Alanine-Scanning Mutagenesis (ASM) are complementary methods that can be used to map the functional importance of amino acid residues within a protein.

Chapter 8: In Vitro Synthesis of Viroids

Viroids are small autonomously replicating RNAs that share structural features with other subviral circular single-stranded RNAs of plants. Viroids and other circular single-stranded RNAs can be synthesised in vitro by a PCR-based procedure using a simple set of reactions. Two end-to-end primers are selected from a desired region of the viroid, one for the synthesis of the first strand cDNA and another for the production of the second strand DNA. The second primer contains an 18 nucleotide T7 promoter at its 5' end, and is selected such that the G nucleotide at the transcription start site represents a G in the viroid. Linked reverse transcription-PCR results in linear double-stranded DNA consisting of the viroid sequence and the T7 promoter. Run-off transcription of the PCR product allows the synthesis of exact- length linear viroid RNA which can be circularised by T4 RNA ligase following an enzymic modification of the 5' triphosphate to a monophosphate. This procedure results in authentic viroid molecules and obviates the need for construction and cloning of DNA in the form of tandem repeats for infectivity tests. It also allows PCR-based manipulation of circular RNAs, thus greatly simplifying structure-function analyses of viroid molecules.

Chapter 9: InVitro Selection of Functional Nucleic Acid Sequences

The power of in vitro selection methods for the isolation of nucleic acids that display a desired property derives from the enormous number of sequence variants that can be surveyed with relative ease using controlled in vitro biochemistry. This methodology has found a variety of applications, ranging from the study of nucleic acid-protein interactions and natural ribozymes to the isolation of nucleic acids with potential as diagnostic or therapeutic reagents or with new catalytic activities. The number of reported applications is growing exponentially, and each application presents new variables and challenges. The goal of this chapter is to guide prospective users through the myriad decisions that must be made in the design and execution of a successful in vitro selection experiment.

Chapter 10: Directed and Random Recombination ff Antibody Sequences

Genetic engineering technologies can be used to clone and produce functional recombinant antibody fragments for various applications, e.g. for basic science and diagnostic applications as well as tumor imaging and targeted therapy. Various PCR techniques can be used to isolate the genes for specific antibodies and also to increase the variability of antigen binding fragments. In this chapter we describe and compare the generation and application of antibody fragments with various degrees of diversity by PCR, using conserved primers (for scFvs and Fabs), by overlap extension PCR (to generate disulfide-stabilized Fv fragments) and by "sexual PCR" for the generation of recombined Fv fragments.

Chapter 11: Construction, Assembly and Selection of Combinatorial Antibody Libraries

Advancements in PCR and phage display technologies have allowed the development of antibody libraries displayed on phage. Although the intact IgG molecule is poorly expressed in bacteria, several different antibody fragments have been produced in large quantities in E. coli (Fabs, Fvs, single-chain Fvs, and diabodies). Combinatorial antibody libraries are constructed using PCR to amplify light chain and heavy chain variable regions. Conserved features of different heavy and light chain families allow PCR primers to be designed to simultaneously amplify many different antibody variable regions from B lymphocyte mRNA. These antibody fragments are expressed as fusion proteins with phage coat proteins. Phage bearing antibody fusion proteins that bind to a given antigen are selected by a process of affinity enrichment called biopanning. Combinatorial antibody libraries have provided an important source of human monoclonal antibodies and allow antibodies to be obtained with or without immunization.

Chapter 12: Beyond the Limits of Natural Diversity: PCR Synthesis of Semi-random Peptide and Antibody Phage Display Libraries

Proteins evolve naturally through mutation followed by selection for activities beneficial to normal functions of organisms. Phage display of random peptide libraries allows the rapid in vitro evolution of polypeptides by selection of high-affinity variants desirable for some artificial objective. While completely random pools of variant proteins might theoretically be an ideal starting point, experimental and biological limitations restrict the diversity of practical libraries. For this reason, randomization is often limited to only five or six codon positions. Although random pentamers and hexamers are useful for the mapping of continuous epitopes recognized by antibodies, these peptides are often too small to be used as ligands for most other proteins. For larger proteins, targeted mutagenesis and semi-random phage display libraries are used to isolate variants with altered or improved specificity and affinity. These approaches are intermediate between random mutagenesis and rational design. Construction of semi-random libraries requires some a priori knowledge of the structural topology and binding domains of the target protein. Specific regions are targeted for randomization using mutagenic PCR primers. Internal random inserts are incorporated by PCR overlap extension and splicing. Several strategies are described for randomization using PCR templates. Specific examples of applications and the design of PCR primers and templates for the construction of semi-random peptide and antibody libraries are discussed.

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