Selection of Specific Cloned DNA in Libraries
Page 770 CLINICAL CORRELATION 18.4 Multiplex PCR Analysis of HGPRTase Gene Defects in Lesch–Nyhan Syndrome Lesch–Nyhan syndrome, as described in Clin. Corr. 12.2, results from a deficiency in hypoxanthine–guanine phosphoribosyltransferase (HGPRTase) activity. Several variant forms of HGPRTase defects have been detected. Multiplex PCR amplification of the HGPRTgene locus has been employed to analyze this gene in cells derived from Lesch– Nyhan patients and results account for the variability of the HGPRTase. The gene, comprised of 9 exons, can be multiplex amplified using 16 different primers in a single PCR. The products can be separated by agarose gel electrophoresis. Analysis of the HGPRTgene locus by multiplex amplification of DNA derived from cells of several patients detected great variations in deletions of different exons to total absence of the exons. Rossiter, B. J. F., et al. In: M. J. McPherson, P. Quirke, and G. R. Taylor (Eds.), PCR. A Practical Approach, Vol. 1. Oxford, England: Oxford University Press, 1994, p. 67. allow one to detect any potential deletion mutation as compared to the normal gene products. Direct sequencing of multiple PCR products can be employed to detect point mutations in the patient gene. Multiplex PCR has been used to detect various defects in the HGPRTase gene in Lesch–Nyhan patients (see Clin. Corr. 18.4). 18.6— Selection of Specific Cloned DNA in Libraries Loss of Antibiotic Resistance Is Used to Select Transformed Bacteria When a single transformed bacterium carrying a recombinant molecule multiplies, its progeny are all genetically the same. If the transformed bacterium carries a recombinant DNA, all progeny will carry copies of the same recombinant plasmid. The foreign DNA has been amplified and is derived from a single cloned DNA fragment. The problem is how to identify the one colony containing the desired plasmid in a field of thousands to millions of different bacterial colonies. The plasmid construct pBR322 and its descendants carry two genes that confer antibiotic resistance. Within these antibioticresistant genes are DNA sequences sensitive to restriction endonuclease. When a fragment of foreign DNA is inserted into a restriction site within the gene for antibiotic resistance, the gene becomes nonfunctional. Bacteria carrying this recombinant plasmid are sensitive to the antibiotic (Figure 18.10). The second antibiotic resistance gene within the plasmid, however, remains intact and the bacteria will be resistant to this antibiotic. This technique of insertional inactivation of plasmid gene products affords a method to select bacteria that carry recombinant plasmids. pBR322 contains genes that confer resistance to ampicillin (ampr) and tetracycline (tetr). A gene library with cellular DNA fragments inserted within the tetr gene can be selected and screened in two stages (Figure 18.10). First, the bacteria are grown in an ampicillincontaining growth medium. Bacteria that are not transformed by a plasmid (they lack a normal or recombinant plasmid) during the construction of the gene library will not grow in the presence of the antibiotic, thus eliminating this population of bacteria. This, however, does not indicate which of the remaining viable bacteria carry a recombinant plasmid vector versus a plasmid with no DNA insert. The second step is to identify bacteria carrying recombinant vectors with nonfunctional tetr genes, which are therefore sensitive to tetracycline. Bacteria insensitive to ampicillin are plated and grown on agar plates containing ampicillin (Figure 18.10). Replica plates can be made by touching the colonies on the original agar plate with a filter and then touching additional sterile plates with the filter. All the plates will contain portions of each original colony at identifiable positions on the plates. The replica plate can contain tetracycline, which will not support the growth of bacteria harboring recombinant plasmids with their tetr gene disrupted. Comparison of replica plates with and without tetracycline will indicate which colonies on the original ampicillin plate contain recombinant plasmids. Thus individual colonies containing the recombinant DNA can be selected, cultured, and analyzed. Either DNA or RNA probes (see pp. 583 and 773) can be utilized to identify the DNA of interest. Ampicillinresistant bacterial colonies on agar can be replica plated onto a nitrocellulose filter and adhering cells from each colony can be lysed with NaOH (Figure 18.10). DNA within the lysed bacteria is also denatured by the NaOH and becomes firmly bound to the filter. A labeled DNA or RNA probe that is complementary to the DNA of interest can be hybridized to the nitrocellulose bound DNA. The filter is exposed to Xray autoradiography. Any colony carrying the cloned DNA of interest will appear as a developed signal on the Xray film. These spots would then correspond to the colony on the Page 771 Figure 18.10 Insertional inactivation of recombinant plasmids and detection of transformed bacteria carrying a cloned DNA of interest. When the insertion of a foreign DNA fragment into a vector disrupts a functional gene sequence, the resulting recombinant DNA does not express the gene. The gene that codes for antibiotic resistance to tetracycline (tetr) is destroyed by DNA insertion while the ampicillin resistance gene (ampr) remains functional. Destruction of one antibiotic resistance gene and retention of a second antibiotic resistance gene allow for the detection of bacterial colonies carrying the foreign DNA of interest within the replicating recombinant vector. Page 772 original agar plate that can then be grown in a largescale culture for further manipulation. Cloned and amplified DNA fragments usually do not contain a complete gene and are not expressed. The DNA inserts, however, can readily be purified for sequencing or used as probes to detect genes within a mixture of genomic DNA, transcription levels of mRNA, and pathological conditions via clinical diagnostic tests. a Complementation for Selecting Bacteria Carrying Recombinant Plasmids Other selection techniques can identify bacteria carrying recombinant DNA molecules. Vectors have been constructed (the pUC series) such that selected bacteria transformed with these vectors carrying foreign DNA inserts can be identified visually (Figure 18.11). The pUC plasmids contain the regulatory sequences and a portion of the 5 end coding sequence (Nterminal 146 amino acids) for the b galactosidase gene (lacZ gene) of the lac operon (Chapter 19, p. 802). The translated Nterminal 146 amino acid fragment of b galactosidase is an inactive polypeptide. Mutant E. coli that code for the missing inactive carboxyterminal portion of b galactosidase can be transformed with the pUC plasmids. The translation of the host cell and plasmid portions of the b galactosidase in response to an inducer, isopropylthio b Dgalactoside, complement each other, yielding an active enzyme. The process is referred to as a complementation. When these transformed bacteria are grown in the presence of a chromogenic substrate (5bromo4chloro3indolyl b Dgalactoside[Xgall) for b galactosidase they form blue colonies. If, however, a foreign DNA fragment is inserted into the base sequence for the Nterminal portion of b galactosidase, Figure 18.11 a Complementation for detection of transformed bacteria. A vector has been constructed (pUC 18) that expresses the Nterminal coding sequence for the enzyme bgalactosidase of the lac operon. Bacterial mutants coding for the Cterminal portion of bgalactosidase are transformed with pUC 18. These transformed bacteria, grown in the presence of a special substrate for the intact enzyme (Xgal), result in blue colonies because they contain the enzyme to react with substrate. The functional Nterminal and Cterminal coding sequences for the gene complement each other to yield a functional enzyme. If, however, a foreign DNA fragment insert disrupts the pUC 18 Nterminal coding sequence for bgalactosidase, bacteria transformed with this recombinant molecule will not produce a functional enzyme. Bacterial colonies carrying these recombinant vectors can then be visually detected as white colonies.