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Degenerate Oligonucleotide Sequence Downloaded from genome.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Degenerate Oligonucleotide Sequence- directed Cross-species PCR Cloning of the BCP 54/ALDH 3 cDNA: Priming from Inverted Repeats and Formation of Tandem Primer Arrays David L. Cooper and Edward W. Baptist The Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710 Bovine corneal protein 54 (BCP 54) these was a 552-bp product occur- PCR (13) to generate the first reported is the major soluble protein of the ring at elevated primer concentra- cDNA probe to BCP 54. (6) The BCP 54 bovine cornea, and immunoreactive tions that formed through bidirec- cDNA probe generated by this mixed forms of this protein have been de- tional amplification from a single oligonucleotide primed amplification scribed in a wide range of mam- DOS annealing to an inverted of cDNA (MOPAC) technique C8,9) was mals. Dideoxy sequence determina- repeat located In the BCP 54 coding cloned and dideoxy sequenced. A Gen- tion of a previously synthesized sequence. The second artifactual Bank library search (version 63.0) 420-bp cDNA to BCP 54 generated product was a 212-bp sequence revealed a strong similarity to the pre- by the novel mixed oligonucleotide that contained several unreported viously cloned cDNA of rat liver (class primer amplification of cDNA amplification anomalies, including 3) tumor-associated aldehyde dehydro- (MOPAC) procedure revealed ex- the formation of a tandem primer genase (RATALD). (12) Nucleotide and tensive similarity to the cDNA en- array. amino acid sequence alignment of the coding tumor-associated rat liver BCP 54 translation product revealed it (class 3) aldehyde dehydrogenase as 78% and 84% identical with (RATALD). PCR amplification with RATALD at the nucleotide and amino additional pairs of degenerate Except for the most abundant soluble acid levels, respectively. Conservation oligonucleotide sequence (DOS) protein of the bovine cornea (BCP 54, of amino acid elements common to primers derived from both BCP 54- molecular weight 54,000 daltons), (1,2) the ALDH supergene family thought to amino-acid sequence and amino little attention has been paid to the be of structural/functional signficance acid and nucleotide sequence data water-soluble structural proteins of the was further substantiated by this analy- from RATALD produced three PCR cornea. Although the initial descrip- sis. products that were cloned and sub- tion of the major soluble corneal Degenerate oligonucleotide se- sequently sequenced. The major protein can be traced back almost 30 quence (DOS) and PCR have been pre- product was 716-bp BCP 54 cDNA years, (3,4) BCP 54 had undergone, until viously utilized successfully to clone clone encompassing the BCP 54 recently (s,6) only minimal molecular cDNAs for specific proteins. This ap- carboxy-terminal amino acid se- characterization with no known func- proach relies on the synthesis of quence for which the DOS pair was tion ascribed. We have previously util- oligonucleotides of limited degeneracy designed. Sequence alignment of ized mixed oligonucleotide primers from reverse translation of protein sub- the BCP 54 cDNA and its transla- complementary to the reverse transla- sequences. The cDNAs so generated tion product with RATALD demon- tion products of amino acid sequence have been used as probes for Northern strated 81% and 85% Identity at obtained from Staphylococcus aureus and Southern blotting and for screen- the nucleotide and amino acid V8-digested BCP 54 fragments (s) and ing cDNA and genomic recombinant levels, respectively. Analysis of the DNA libraries. (8,9) DOS primers have additional two clones established Present address: Department of Pathology, University also been used to amplify unknown that they were the results of PCR of Pittsburgh School of Medicine, Pittsburgh, members of gene families (1~ and artifactual processes. The first of Pennsylvania 15261. homologous genes in different 1:57-629 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/91 $3.00 PCR Methods and Applications 57 Downloaded from genome.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press species. (11) These applications of DOS incubation at 72~ followed to insure BCP 54 cDNA that encompasses the 3' have become common, but little has completion of all polymerization prod- most terminal protein encoding region been reported concerning the composi- ucts. Aliquots (10 ~l) of reaction mix- as well as two minor products that tion and nature of inappropriate prod- tures were analyzed by electrophoresis were generated during amplification. ucts generated by PCR with such through 1.2% agarose gels. Following Our strategy utilizes single-stranded primers. In this report we communi- with ethidium bromide and photo- (ss) bovine corneal epithelial cDNA as cate not only the successful genera- graphed with UV illumination. template for PCR amplification primed tion, again by the MOPAC technique, A second set of experiments by two DOS (Fig. 1) based on pre- of a second extended 716-bp BCP 54 employed identical reaction condi- viously determined BCP 54 and RAT- cDNA that encompasses the 3' most tions, except that 400 ng each of ALD amino acid and nucleotide se- terminal protein-encoding region, but primers DOS#1 and DOS#2 or DOS#1 quence. (s,12) The 5' sense primer additionally discuss the nature of two alone were used. This reproducibly (DOS#l) and an internal reverse primer PCR-generated artifacts associated with generated the 552-bp band that we had (DOS#2) were previously designed (6) by the utilization of DOS as PCR primers. previously observed. (6) reverse translation of amino acid se- quence determined from a BCP 54 V8 METHODS AND MATERIALS Cloning of PCR Products protease fragment (Fig. 2A). The 3' antisense primer (DOS#3) was Materials Reaction mixtures with both the 716- bp and 552-bp bands were extracted designed (16) from the 3' nucleotide PCR primers DOS#1 (AACCCYCAYTA- with CHC13 after most of the mineral and amino acid sequence of the rat YGTGGACAARGA) and DOS#2 (GAAC- oil was removed by pipetting. The homolog RATALD. Based on dinucleo- TGRATRGCYTCYTC) were designed as DNA was precipitated with ethanol, tide frequency utilization in human previously described C6) by reverse trans- and the pellet was dissolved in 10 ~l of proteins, (17) the 3' sequence syn- lation of amino-terminal amino acid 10 mM Tris-HCl (pH 8.5), 1 mM EDTA. thesized consisted of the singular sequences of BCP 54 peptides produced After restriction enzyme digestion methionine codon, the most frequent- by partial digestion with Staphylococcus with EcoRI, the PCR product was ly utilized proline codon, the two most aureus V8 protease. Primer DOS#3 (GT- ligated into plasmid pBS- which had frequent codons of asparagine and GYCKGGGCATRTTRGC) was similarly been previously digested with EcoRI alanine, and four of six possible prepared from reverse translation of and treated with bacterial alkaline codons of arginine. We also designed a the carboxy-terminal 6 amino acids of phosphatase. Transformation of com- fourth DOS primer to the 5 ' terminus the RATALD sequence ~12/ (Fig. 2A). petent Escherichia coli strain NM522 (is) of RATALD and paired it with our suc- Each degenerate oligonucleotide also with the ligation reaction mixture cessful DOS#2 and DOS#3 as well as a contained an 8-nucleotide linker on its resulted in a number of white colonies number of other internal BCP 54 5' terminus that included a 6-nucleo- on ampicillin (100 mg/ml) IPTG (0.5 cDNA-based nondegenerate synthetic tide EcoRI restriction site (GGGAA- mM) XGAL (40 mg/ml) LB solid me- oligonucleotide primers. These at- TTC). Restriction enzymes and Taq dium. Twelve of these were picked, tempts failed to amplify the 5' DNA polymerase were purchased from restreaked, and analyzed by agarose gel terminus. We believe this may indicate Promega (Madison, WI), and T4 DNA electrophoresis of alkaline lysis DNA the incomplete nature of 5' ends con- ligase and bacterial alkaline phospha- minipreparations digested with EcoRI. tained in our ss cDNA preparation used tase from GIBCO BRL/Life Tech- in this work, and not necessarily an nologies (Gaithersburg, MD). Plasmid Sequence Analysis alternative 5 ' bovine sequence. pBS- was obtained from Stratagene (La Plasmid DNA from colonies containing Plasmid DNA from clone #6 which Jolla, CA). recombinant plasmids was used as contained a PCR-generated insert of template in double-stranded dideoxy approximately 720 bp (Fig. 2B) was PCR Amplification sequencing reactions with T7 DNA used as template in double-stranded The first strand cDNA synthesized from polymerase kits from either Pharmacia dideoxy sequencing reactions bovine corneal epithelium mRNA was or US Biochemical. By sequencing from (Pharmacia or US Biochemical). By se- used directly in 50-~1 PCR reac- primer sites on both sides of the pBS- quencing from T3/T7 promoter sites tions ~13,14J which contained 0.2 mM of polylinker, and the generation of inter- on both sides of the pBS- polylinker, each dNTP, 200 ng each of primers nal deoxyoligonucleotide primers and utilizing two internal synthetic DOS#1 and either DOS#2 or DOS#3, homologous to the cDNA sequence, oligonucleotides made to previous BCP and 2 units of Taq DNA polymerase in the full sequence of the PCR cDNA was 54 cDNA sequence, the full sequence a buffer of 10 mM Tris-HC1 (pH 9.0 at obtained. To control for Taq DNA of the PCR cDNA was obtained (Fig. 3). 25~ mM KC1/1.5 mM MgC12/0.1% polymerase misincorporations, DNA The cDNA contains 716 nucleotides, gelatin/0.01% Triton X-IO0. Reactions from 10 independent recombinant iso- or, when translated, 239 amino acids. were overlaid with 50 ~l of mineral oil lates identical to clone #6 as described Sequence alignment of the BCP 54 to prevent evaporation and heated at in the text were combined and se- cDNA is colinear with the RATALD se- 94~ for 4 min to denature initially quenced as above.
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