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Cloning Gene Family Members 485 67 Cloning Gene Family Members Using PCR with Degenerate Oligonucleotide Primers Gregory M. Preston 1. Introduction 1.1. What Are Gene Families? As more and more genes are cloned and sequenced, it is apparent that nearly all genes are related to other genes. Similar genes are grouped into families, such as the collagen and globin gene families. There are also gene superfamilies. Gene superfamilies are composed of genes that have areas of high homology and areas of high divergence. Examples of gene superfamilies include the oncogenes, homeotic genes, and myosin genes. In most cases, the different members of a gene family carry out related functions. A detailed protocol for the cloning by degenerate oligonucleotide polymerase chain reaction (PCR) of members of the Aquaporin family of membrane water channels (1,2) is discussed here. 1.2. Advantages of PCR Cloning of Gene Family Members There are several considerations that must be taken into account when determining the advantages of using PCR to identify members of a gene family over conventional cloning methods of screening a library with a related cDNA, a degenerate primer, or an antibody. It is recommended that after a clone is obtained by PCR, one uses this template to isolate the corresponding cDNA from a library because mutations can often be introduced in PCR cloning. Alternatively, sequencing two or more PCR clones from independent reactions will also meet this objective. The following is a list of some of the advantages of cloning gene family members by PCR. 1. Either one or two degenerate primers can be used in PCR cloning. When only one of the primers is degenerate, the other primer must be homologous to sequences in the phage or bacteriophage cloning vector (3,4) or to a synthetic linker sequence, as with RACE PCR. The advantage to using only one degenerate primer is that the resulting clones contain all of the genetic sequence downstream from the primer (be it 5′ or 3′ sequence). The disadvantage to this anchor PCR approach is that one of the primers is recognized by every gene in the starting material, resulting in single-strand amplification of all sequences. This is particularly notable when attempting to clone genes that are not abundant in the starting material. This disadvantage can often be ameliorated in part by using a nested From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ 485
486 Preston amplification approach with two degenerate primers to preferentially amplify desired sequences. 2. It is possible to perform a PCR on first-strand cDNAs made from a small amount of RNA and, in theory, from a single cell. Several single-stranded “minilibraries” can be rapidly prepared and analyzed by PCR from a number of tissues at different stages of development or cell cultures under different hormonal conditions. Therefore, PCR cloning can potentially provide information about the timing of expression of an extremely rare gene family member, or messenger RNA splicing variants, that may not be present in a recombinant library. 3. Finally, the time and expense required to clone a gene should be considered. Relative to conventional cloning methods, PCR cloning can be more rapid, less expensive, and in some cases, the only feasible cloning strategy. It takes at least 4 d to screen 300,000 plaques from a λgt10 library. With PCR, an entire library containing 10 8 independent recombinants (~5.4 ng DNA) can be screened in one reaction. Again, to ensure authenticity of your PCR clones, you should either use the initial PCR clone to isolate a cDNA clone from a library or sequence at least two clones from independent PCRs. 1.3. Degenerate Oligonucleotide Theory and Codon Usage Because the genetic code is degenerate, primers targeted to particular amino acid sequences must also be degenerate to encode the possible permutations in that sequence. Thus, a primer to a six- amino acid sequence that has 64 possible permutations can potentially recognize 64 different nucleotide sequences, one of which is to the target gene. If two such primers are used in a PCR, then there are 64 × 64 or 4096 possible permutations. The target DNA will be recognized by a small fraction (1/64) of both primers, and the amplification product from that gene will increase exponentially. However, some of the other 4095 possible permutations may recognize other gene products. This disadvantage can be ameliorated by performing nested amplifications and by using “guessmer” primers. A guessmer primer is made by considering the preferential codon usage exhibited by many species and tissues (see Subheading 3.1.). For instance, the four codons for alanine begin with GC. In the third position of this codon, G is rarely used in humans (~10.3% of the time) or rats (~8.0%), but often used in Escherichia coli (~35%) (5). This characteristic of codon usage may be advantageously used when designing degenerate oligonucleotide primers. 1.4. Strategy for Cloning Aquaporin Gene Family Members In a related methods chapter (3), I described the cloning by degenerate primer PCR of Aquaporin-1 (formerly CHIP28) from a human fetal liver λgt11 cDNA library starting with the first 35 amino acids from the N-terminus of the purified protein. A full-length cDNA was subsequently isolated from an adult human bone marrow cDNA library (4) and following expression in Xenopus shown to encode a water selective channel (6). We now know that the Aquaporin family of molecular water channels includes genes expressed in diverse species, including bacteria, yeast, plants, insects, amphibians, and mammals (1,2,7). We have used degenerate oligonucleotide primers designed to highly conserved amino acids between the different members of the Aquaporin family to clone novel Aquaporin gene family cDNAs from rat brain (AQP4) and salivary gland (AQP5) libraries (8,9). In Subheading 3., I will describe the creation of a new set of degenerate primers that we have used to clone, by degenerate primer PCR, Aquaporin homologs from a number of different tissues and species. Subheading 3.
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Page 483 and 484: 496 Preston MgCl 2 concentrations b
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Page 513 and 514: 526 Burke and Barik Fig. 1. The bas
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