Published online January 29, 2004 Nucleic Acids Research, 2004, Vol. 32, No. 2 e22 DOI: 10.1093/nar/gnh021 Oligonucleotide-directed site-speci®c integration of high complexity libraries into ssDNA templates M. B. Hale1,2,3, G. P. Nolan2,3,* and R. Wolkowicz2,3 1Department of Molecular Pharmacology, 2Department of Microbiology and Immunology and 3Baxter Laboratory in Genetic Pharmacology, School of Medicine, Stanford University, Stanford, CA 94305, USA Received October 31, 2003; Revised and Accepted December 22, 2003 ABSTRACT cassette cloning approach. In brief, a small oligonucleotide complementary to the non-random 3¢ end of the library We present an approach that generates an oligomer- oligonucleotide is annealed to prime a polymerase reaction Downloaded from based library with minimal need for restriction site that makes the library insert double stranded (3). The now modi®cation of sequences in the target vector. The double-stranded insert is restricted with endonucleases, puri- technique has the advantage that it can be applied ®ed by gel electrophoresis and ligated into a vector previously for generating peptide aptamer libraries at sites digested with complementary restriction enzymes. Because within proteins without the need for introducing the oligonucleotide is usually less than 100 bases long, it can ¯anking enzyme sites. As an example we present a be dif®cult to ef®ciently purify the double-stranded insert that nar.oxfordjournals.org phagemid retroviral shuttle vector that can be used was successfully cut with both restriction enzymes from to achieve stable expression of the library in mam- incompletely digested material. Both the ligation of a small malian cells for the purpose of screening for pep- insert into a much larger vector and the inability to adequately tides with desired biological activity. purify the insert can result in loss of library complexity. Here we have considered a different strategy: the creation of a single-stranded backbone vector that is compatible with a INTRODUCTION single-stranded insert containing the aptamer library. at LibrarySerials on September 14, 2010 Although such an approach has previously been used primar- A number of cloning strategies have evolved over the past ily for the substitution or incorporation of one or a few three decades ranging from the conventional cassette cloning nucleotides, we were encouraged that such site-directed approach using restriction enzymes and ligase to more recent mutagenesis has been used to successfully integrate sequences techniques involving cre recombinase (1) and integrase± as large as 27 bases such as the HA1 epitope (15), a size equal exisionase systems (2). The latter techniques are adequate for to that of many libraries. However, conventional site-directed basic cloning where the goal is to transfer a single insert into a mutagenesis is an inef®cient process that yields the desired vector. However, these techniques are less powerful when the number of unique sequences to be inserted increases. Such is product much less than 50% of the time (16), an ef®ciency too the case with aptamer libraries that require the insertion of low for library generation of suf®cient complexity. The more thousands or millions of different sequences into the same advanced QuikChange Mutagenesis method is still incapable backbone vector. of introducing sequences long enough to generate biologically At the DNA level an aptamer library can be de®ned by a active peptide libraries. When a 31 nucleotide sequence was constant region shared by all clones and a variable region that introduced, more than 25% of the transformants failed to carry is unique for every member represented in the library. In order the insert even after substantial optimization (17). This to achieve a high complexity library, it is necessary to insert a procedure does not improve transformation ef®ciency, critical suitably large number of unique inserts into a speci®c site for complex library production. within the vector. This is applicable for peptide libraries and The technique presented here uses a library oligonucleotide ribozyme libraries, among others (3±10). The number of that hybridizes to the single-stranded vector, and primes a unique clones within the library de®nes its complexity, and it polymerase reaction that uses the vector strand as template. is usually desirable to have a high complexity that represents The newly synthesized library strand is covalently closedÐ as many different sequences as possible. The creation of such creating a double-stranded DNA (dsDNA)Ðand puri®ed from libraries can represent a signi®cant share of the time invested template materials. Modi®cations to the technique ensure that in setting up a genetic screen using such libraries. nearly 100% of the resulting vectors can contain inserts. We The construction of a random peptide expression library demonstrate that the procedure is suf®ciently ef®cient to requires a random central region usually 27±45 nucleotides in generate libraries of a complexity of at least 1 3 106. With length ¯anked by regions of de®ned sequence and the optimization and increases in scale it should be possible to backbone vector chosen to carry the library. Examples of make libraries of 1 3 108. The approach should simplify the such libraries can be found in multiple publications (3,5,11± creation of high complexity oligomer-based libraries in a 14). The process usually involves a modi®ed version of the number of experimental settings. *To whom correspondence should be addressed. Tel: +1 650 725 7002; Fax: +1 650 725 2383; Email: [email protected] Nucleic Acids Research, Vol. 32 No. 2 ã Oxford University Press 2004; all rights reserved e22 Nucleic Acids Research, 2004, Vol. 32, No. 2 PAGE 2 OF 7 Transformation of the heteroduplex MATERIALS AND METHODS The heteroduplex product was puri®ed by agarose gel Strains electrophoresis as the band that co-migrates with the covalently closed circular band of the double-stranded vector The Escherichia coli strain XL1-Blue was used in this study and was excised and phenol extracted. One microliter of the for transformation of plasmids and production of phage. This puri®ed DNA was used to transform 20 ml of electrocompetent strain carries an F' episome that confers tetracycline resistance XL1-Blue E.coli per electroporation cuvette (0.1 cm). and is required for pilus formation and phage infection. After rescuing for 2 h at 32°C in salt-optimized + carbon M13KO7 bacteriophage was used as the helper phage. (SOC) broth with vigorous agitation the entire transformation was used to innoculate 20 ml of LB containing ampicillin Puri®cation of ssDNA template (100 mg/ml). In addition, a small quantity of the transform- Bacteria transformed with the phagemid were cultured in LB ation was plated on ampicillin plates to determine library containing ampicillin (100 mg/ml) + tetracycline (50 mg/ml). complexity. The liquid culture was grown overnight and the This culture inoculated 2YT containing ampicillin (100 mg/ml) plasmid DNA puri®ed by alkaline lysis followed by binding to and helper phage. After 2 h of helper phage exposure, silica columns±Qiagen's Miniprep kit. kanamycin (50 mg/ml) was added and the culture incubated at Elimination of the template strand Downloaded from 32°C with agitation overnight. At stationary phase the bacteria were pelleted, the supernatant spun twice and ®ltered through One microgram of the plasmid prep was digested with SphI as a 0.45 mm Acrodisc ®lter. One hundred and ®fty microliters of above, visualized by agarose gel electrophoresis, and used to a 20% solution of PEG 8000 and 2.5 M NaCl was added per transform 20 ml of electrocompetent XL1-Blue E.coli. After milliliter of supernatant and incubated at 4°C for 45 min to rescuing for2hat32°C in SOC with vigorous agitation the precipitate the phage. The mixture was centrifuged at 11 000 g transformation was used to innoculate 20 ml of LB containing nar.oxfordjournals.org to pellet the phage. The pellet was resuspended in EDTA and ampicillin (100 mg/ml). After incubation overnight at 32°C the single-stranded DNA (ssDNA) extracted by phenol followed culture was either prepped directly or used to innoculate by precipitation with sodium acetate and ethanol. A 250 ml 500 ml of ampicillin±LB. culture grown overnight at 32°C with agitation yielded ~2 mg of ssDNA. RESULTS AND DISCUSSION Library strand synthesis We outline the approach by which we generate oligonucleo- at LibrarySerials on September 14, 2010 The library oligonucleotide (50 pmol) and 2 mg of the ssDNA tide-directed site-speci®c libraries as follows: (i) puri®cation template was combined with 0.5 ml of `Platinum Taq High of a single-stranded phagemid acceptor vector; (ii) design of a Fidelity' (a mixture of heat-activated Taq polymerase and corresponding library oligonucleotide insert ssDNA; (iii) Pyrococcus species GB-D polymerase from Invitrogen) in extension of the library ssDNA by priming; (iv) covalent 200 mM dNTP, 2 mM MgSO , 60 mM Tris±SO (pH 8.9), closing of the extended library strand to create a dsDNA for 4 4 transformation; and (v) puri®cation of the double-stranded 180 mM ammonium sulfate. This enzyme cocktail was chosen library (see Figure 1 for outline of protocol). to minimize misincorporation and enzyme stalling. It was activated by incubation at 95°C for 50 s followed by ramping Phagemid and oligonucleotide design down to 60°C over 1 min to anneal the library oligonucleotide. This technique required the puri®cation of one of two potential The sample temperature was then increased to 68°C for circular single strands of the vector used to express the library. 30 min. The reaction was quenched by addition of buffer- We created a phagemid that contained both the E.coli origin of saturated phenol to minimize low temperature mispriming.
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