A cloning vehicle suitable for strand separation

A new plasmid has been constructed which contains a poly(A) : poly(dT) duplex segment of length approx. 100 base pairs (bp) inserted into the PvuII site of pBR322. This plasmid, pKH47, has all the other restriction sites of pBR322 available for insertion of foreign DNA, and has the same drug resistance genes as does the parental plasmid. The complementary strands of the linearized denatured plasmid DNA can be separated rapidly an efficiently by affinity chromatography with oligo(dA)- and oligo(dT)-cellulose columns in series. More than 90% of the input DNA is recovered as separated strands which can be annealed to form full length double-stranded molecules. One of the applications of the plasmid is to prepare separated complementary strands for sequencing by the chain-terminator technique using DNA primers. This application is illustrated by a sequencing example for a Drosophila DNA insert carrying a tRNA gene.     

Single-stranded poly(deoxyguanylic acid) associates into double- and triple-stranded structures

Circular plasmid deoxyribonucleic acid (DNA), pBR322, was digested with the restriction endonuclease PstI to give full-length double-stranded DNA molecules, terminated by two self-complementary single-stranded sequences: (formula: see text). The protruding 3' termini were extended with dG by using calf thymus terminal deoxynucleotidyl transferase and dGTP, to form single-stranded tails of oligo(dG). At a length of about dG15, such tails become resistant to single strand specific endonuclease S1, and also cease to function as substrate (initiator) for the terminal deoxynucleotidyl transferase. This altered reactivity arises from association of the oligo(dG) tails into double- and triple-stranded structures, resulting in linear, circular, and branched polymers of the monomeric linear plasmid DNA. All these polymeric structures of the plasmid DNA are stable at room temperature, can be observed in the electron microscope, and can be separated from each other by agarose gel electrophoresis. At 60 degrees C or in 50% formamide, most of the oligo(dG) self-association can be reversed (melted), and the plasmid DNA is again found as the original linear monomer.     

A stable complex between homopyrimidine oligomers and the homologous regions of duplex DNAs

When plasmid DNA duplexes carrying the regular homopurine-homopyrimidine inserts (dGA)n, (dTC)n and (dG)n, (dC)n are preincubated with homologous labeled oligo(dPy) ((dTC)n and (dC)n respectively) at acid pH, the label co-electrophoreses with the duplex DNA. Thus, a very strong complex is formed. Complementary oligo(dPu) does not form a complex under these conditions. No binding is observed for oligo(dPy) with non-homologous inserts as well as with vector plasmids without inserts. The complex is formed equally well with linear, nicked or superhelical DNA. The complex is not detected at pH greater than 6. Complex formation leads to very little, if any, unwinding of the duplex. The observed complex appears to be the Py.Pu.Py triplex consisting of TAT and CGC base-triads with protonated cytosines. Two-dimensional gel electrophoresis patterns show that the presence of homologous oligo(dPy) destabilizes the formation of the H form.     

Resolution of sequencing ambiguities: a universal FokI adapter permits Maxam-Gilbert re-sequencing of single-stranded phagemid DNA

We propose a method to resolve ambiguities encountered when single-stranded (ss) phagemid DNA templates are sequenced by the dideoxy method. A single oligodeoxyribonucleotide (oligo) is synthesized with the following features: (i) the 20 nucleotides (nt) at the 5'-end form a double-stranded hairpin containing a FokI restriction site, exactly as previously described by Podhajska and Szybalski [Gene 40 (1985) 175-182]; (ii) the 23 nt at the 3'-end hybridize to the (+)strand of ss phagemid DNA in the region complementary to the M13 universal sequencing primer. In a simple one-tube set of reactions, ss phagemid DNA is annealed to this oligo, cleaved by FokI at a unique site outside the vector multiple cloning site and then labelled at this unique site by Klenow polymerase and [alpha-32P]dCTP. These reactions provide a convenient route by which Maxam-Gilbert chemical degradation sequencing methods can be used to resolve ambiguities encountered in the dideoxy-sequencing of a unidirectional deletion series already prepared in popular phagemid vectors. A single oligo allows labelling of all members of a deletion series. A second universal oligo allows the same set of reactions to be applied to inserts cloned into (-)strand phagemids.     

Molecular cloning of DNA complementary to a mouse α-fetoprotein mRNA sequence

DNA complementary to mouse yolk sac messenger RNA has been inserted at the PstI site of the plasmid pBR322 by annealing of the oligo(dG)-tailed plasmid DNA with the oligo(dC)-tailed mouse DNA. Transformation of Escherichia coli strain RRI with this annealed DNA yielded clones bearing recombinant plasmids. The clones were screened for DNA complementary to mouse a-fetoprotein (AFP) messenger RNA sequences by hybridization with a cDNA probe transcribed from an AFP mRNA of over 90% purity. Out of nine plasmids that were isolated and analyzed by restriction mapping, all had homologous insert DNA of various lengths. The plasmid with the longest insert, pAF6, contained 1.65 kb of added DNA, which is about 70% of the AFP mRNA. This clone was positively identified by a hybridization-translation procedure to contain a cDNA sequence for AFP. A restriction map of this clone and the orientation of the message are presented.     

Isolation of oligo(U)-containing heterogeneous nuclear RNA from control and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-treated HeLa cells

Most (54–79%) of the heterogeneous nuclear RNA (hnRNA) which contains oligo(U) sequences was specifically retained on columns of poly(A) Sepharose and separated from hnRNA which lacked oligo(U) sequences. The isolation of oligo(U)-containing hnRNA was maximized by treating the hnRNA with HCHO prior to chromatography. This permitted an initial characterization of the oligo(U)-containing hnRNA. Experiments suggest that HCHO denatured the hnRNA and rendered the oligo(U) sequences accessible to poly(A) Sepharose. In denatured hnRNA, the percentage of molecules which contained an oligo(U) sequence increased with the size of the hnRNA; 32–57% of the large hnRNA [8–13 kilobases (kb) long] contained an oligo(U) sequence while only 11–14% of the 2-kb-long hnRNA contained an oligo(U) sequence. The number of oligo(U) sequences per molecule was also measured in denatured hnRNA of varying length. While the largest hnRNA class analyzed (13 kb) was found to contain the highest percentage of oligo(U)-containing molecules (57%), the 8- and 2-kb-long hnRNA fractions contained a greater total number of oligo(U)-containing molecules. The percentage of hnRNA molecules which contained an oligo(U) sequence, the number of oligo(U) sequences per molecule, and the size of the oligo(U) sequence were similar in both control hnRNA and the fraction of hnRNA (~30%) which is resistant to inhibition by 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole.     

Sequence-specific chemical modification of double-stranded DNA with alkylating oligodeoxyribonucleotide derivatives

Chemical modification of double-stranded (ds) DNA with alkylating oligodeoxynucleotide (oligo) derivatives, 5'-p(N-2-chloroethyl-N-methylamino) benzylamides of oligos, has been investigated. In contrast to relaxed plasmid DNAs, the superhelical molecules interact with the oligo derivatives and specific alkylation of the DNAs occurs at the regions complementary to the oligo reagents. Alkylating derivatives of oligocytidylates and pT(pCpT)6 react with corresponding homopyrimidine-homopurine tracts within ds DNA fragments due to triple helix formation.     

Specificity of the nick-closing activity of bacteriophage T4 DNA ligase

Bacteriophage T4 DNA ligase effectively joins two adjacent, short synthetic oligodeoxyribonucleotides (oligos), as guided by complementary oligo, plasmid and genomic DNA templates. When a single bp mismatch exists at either side of the ligation junction, the efficiency of the enzyme to ligate the two oligos decreases. Mismatch ligation is approximately five-fold greater if the mismatch occurs at the 3' side rather than at the 5' side of the junction. During mismatch ligation the 5' adenylate of the 3' oligo accumulates in the reaction. The level of the adenylate formation correlates closely with the level of the mismatch ligation. Both mismatch ligation and adenylate formation are suppressed at elevated temperatures and in the presence of 200 mM NaCl or 2-5 mM spermidine. The apparent Km for the oligo template in the absence of salt is 0.05 microM, whereas the Km increases to 0.2 microM in the presence of 200 mM of NaCl. In this report, we demonstrate these properties of T4 DNA ligase for oligo pairs complementary to the beta-globin gene at the sequence surrounding the single bp mutation responsible for sickle-cell anemia. Because of the highly specific nature of the nick-closing reaction, ligation of short oligos with DNA ligase can be used to distinguish two DNA templates differing by a single nucleotide.     

Selection of oligonucleotide probes for protein coding sequences

MOTIVATION: Large arrays of oligonucleotide probes have become popular tools for analyzing RNA expression. However to date most oligo collections contain poorly validated sequences or are biased toward untranslated regions (UTRs). Here we present a strategy for picking oligos for microarrays that focus on a design universe consisting exclusively of protein coding regions. We describe the constraints in oligo design that are imposed by this strategy, as well as a software tool that allows the strategy to be applied broadly. RESULT: In this work we sequentially apply a variety of simple filters to candidate sequences for oligo probes. The primary filter is a rejection of probes that contain contiguous identity with any other sequence in the sample universe that exceeds a pre-established threshold length. We find that rejection of oligos that contain 15 bases of perfect match with other sequences in the design universe is a feasible strategy for oligo selection for probe arrays designed to interrogate mammalian RNA populations. Filters to remove sequences with low complexity and predicted poor probe accessibility narrow the candidate probe space only slightly. Rejection based on global sequence alignment is performed as a secondary, rather than primary, test, leading to an algorithm that is computationally efficient. Splice isoforms pose unique challenges and we find that isoform prevalence will for the most part have to be determined by analysis of the patterns of hybridization of partially redundant oligonucleotides. AVAILABILITY: The oligo design program OligoPicker and its source code are freely available at our website.     

Novel route to oligo(deoxyribonucleoside phosphorothioates). Stereocontrolled synthesis of P-chiral oligo(deoxyribonucleoside phosphorothioates).

The synthesis and separation of diastereoisomerically pure 5'-O-DMT-nucleoside 3'-O-(2-thio-1,3,2-oxathiaphospholane) allows their use as synthons in DBU-catalyzed reaction with the 5'-hydroxyl function of solid-support-bound nucleoside moiety. Since this reaction is stereospecific (greater than 99%), this novel method allows preparation of oligo(nucleoside phosphorothioates) with predetermined chirality at each P-chiral internucleotide phosphorothioate centre.     

The identification of cDNA clones that include the 3′ end of mRNA

A method is presented that facilitates the identification of cDNA clones corresponding to the polyadenylated 3′ end of mRNA. It is based on the use of a poly dT probe that is synthesized by homopolymer extension of commercially available oligo dT. The method is shown to work in Southern blot analysis of plasmid preparations and in situ with colonies.     

Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering

Recombination with single-strand DNA oligonucleotides (oligos) in Escherichia coli is an efficient and rapid way to modify replicons in vivo. The generation of nucleotide alteration by oligo recombination provides novel assays for studying cellular processes. Single-strand exonucleases inhibit oligo recombination, and recombination is increased by mutating all four known exonucleases. Increasing oligo concentration or adding nonspecific carrier oligo titrates out the exonucleases. In a model for oligo recombination, λ Beta protein anneals the oligo to complementary single-strand DNA at the replication fork. Mismatches are created, and the methyl-directed mismatch repair (MMR) system acts to eliminate the mismatches inhibiting recombination. Three ways to evade MMR through oligo design include, in addition to the desired change (1) a C·C mismatch  6 bp from that change; (2) four or more adjacent mismatches; or (3) mismatches at four or more consecutive wobble positions. The latter proves useful for making high-frequency changes that alter only the target amino acid sequence and even allows modification of essential genes. Efficient uptake of DNA is important for oligo-mediated recombination. Uptake of oligos or plasmids is dependent on media and is 10,000-fold reduced for cells grown in minimal versus rich medium. Genomewide engineering technologies utilizing recombineering will benefit from both optimized recombination frequencies and a greater understanding of how biological processes such as DNA replication and cell division impact recombinants formed at multiple chromosomal loci. Recombination events at multiple loci in individual cells are described here.     

FokI method of gene synthesis

An accurate, fast and simple method is presented for synthesis of a gene, or any DNA fragment with a defined sequence. The method is based on the observation that large (approx. 100 bp long) inserts can be cloned into a plasmid using a technique of oligodeoxynucleotide (oligo)-directed double-strand (ds) break repair. The procedure involves transformation of Escherichia coli with a denatured mixture of an insert-carrying oligo and linearized plasmid DNA [Mandecki, Proc. Natl. Acad. Sci. USA 83 (1986) 7177-7181]. The nucleotide (nt) sequences are inserted between two FokI restriction nuclease sites in one of four pUC-derived plasmids. Since FokI makes a staggered ds break at a DNA site 9 and 13 nt away from its recognition site, upon cleavage of the plasmid DNA with FokI, a restriction fragment is liberated that by design contains unique 4-nt-long 5'-protruding ends. The uniqueness of ends permits efficient and directed simultaneous ligation of several restriction fragments to form a gene. The method offers flexibility due to the modular-type assembly and does not require any restriction sites within the constructed gene. The sequence error rate is low: about one error per 4000 bp of DNA cloned. Synthetic DNA for only one DNA strand needs to be provided. The method was applied to the synthesis of a gene fragment encoding the N-terminal 143 amino acid residues of the human immunodeficiency virus transmembrane protein (p41).