Chander P. Bahl

A general method for inserting specific DNA sequences into cloning vehicles

A general method has been developed to introduce any double-stranded DNA molecule into cloning vehicles at different restriction endonuclease sites. In this method a chemically synthesized decadeoxyribonucleotide duplex, containing a specific restriction endonuclease sequence, is joinlex DNA is cut by the same restriction endonuclease to generate the cohesive ends. It is then inserted into the restriction endonuclease cleavage site of the cloning vehicle. To demonstrate the feasibility of this new method, we have inserted separately the synthetic lac operator DNA at the Bam I and HindIII cleavage sites of the plasmid pMB9 DNA.

A rellable mapping method for sequence determination of oligodeoxyribonucleotides by mobility shift analysis

Abstract The method for sequence analysis of large oligodeoxyribonucleotides based on the characteristic mobility shifts of their sequential partial degradation products on two-dimensional homochromatography has been perfected using a large number of synthetic oligodeoxyribonucleotides of defined sequences as standards. Flat bed electrophoresis with careful temperature control gave entirely reproducible mobilities in the first dimension. Using this information, an accurate formula has been derived for calculating the relative electrophoretic mobilities of oligodeoxyribonucleotides of any composition. This formula is used to calculate the mobility shifts between two consecutive oligodeoxyribonucleotides in a series of partial products of an unknown oligomer distributed in the two-dimensional homochromatogram which differ by one nucleotide in length. This is compared with the observed mobility shift value to identify the added nucleotide. This provides a direct and rapid method for obtaining the unambiguous sequence of an entire oligodeoxyribonucleotide up to 15 nucleotides in length.

[7] Synthetic adaptors for cloning DNA

Publisher Summary This chapter discusses synthetic adaptors for cloning deoxyribonucleic acid (DNA). Chemically synthesized oligonucleotides that contain restriction-enzyme-recognition sequences are useful tools for molecular biologists. Three major types of adaptor oligonucleotides have been synthesized for increasing flexibility in molecular cloning: a self-complementary oligomer, which contains a restriction-endonuclease-recognition site, ready-made adaptors, and conversion adaptors. The most flexible and general use of synthetic adaptors is cloning a large variety of DNA fragments. For this approach, blunt-ended adaptors are first ligated onto the termini of a blunt-ended DNA fragment and subsequently cut with the appropriate restriction enzyme to generate cohesive ends. This adapted DNA can then be cloned easily. Synthetic adaptors can also be used to introduce new restriction sites into cloning vectors, thereby augmenting their versatility. This chapter describes the methodology used to insert various restriction sites into the phage vector M13mp2 and the Escherichia coli ( E. coli ) plasmid pBR322 and demonstrates the use of a combination of two different blunt-ended adaptors and the scheme by which conversion adaptors can be utilized for increasing flexibility in molecular cloning. Concepts related to the introduction of cohesive ends at the termini of DNA are discussed in this chapter..

Chemical synthesis of versatile adaptors for molecular cloning

Abstract In this communication we report the chemical synthesis of two types of oligodeoxynucleotides to be used as adaptors in molecular cloning. The first type is used to create specific cohesive end sequences at the termini of a blunt-end DNA molecule without the use of restriction enzymes. The second type of adaptor is used to convert one kind of restriction-enzyme-generated specific cohesive end to another. This includes both the conversion of one type 5′-protruding end to another 5′-protruding end, and of a 3′- to a 5′-protruding end and vice versa.

Synthetic oligodeoxynucleotides for analyses of DNA structure and function.

Publisher Summary This chapter discusses that synthetic oligonucleotides plays a significant role in understanding of many current problems in molecular biology. Synthetic homopolymeric oligodeoxynucleotides have been extensively used for studying the mechanism of action of various enzymes on Deoxyribonucleic acid (DNA) synthesis in vitro. Synthesis of all of the possible 64 triribonucieoside diphosphates has been accomplished for the elucidation of the genetic code. It reviews that several segments of DNA possessing important biological functions have been chemically synthesized. These include two transfer ribonucleic acid (RNA) genes, an RNase S-peptide gene, an angiotensin hormone gene, several lactose operator DNA segments, and several restriction endonuclease recognition sequences. The availability of these synthetic molecules has given us the opportunity to understand these systems in greater depth. Short segments of oligodeoxynucleotides have also been synthesized as DNA primers for sequence analysis. The chapter presents a general picture of how synthetic oligonucleotides have been used to solve various problems in molecular biology. It concludes that, optimal conditions may be found so that large quantities of this RNA transcript would then be used to direct the synthesis in the micro-organism of this protein product in bulk quantities. The potential for large-scale production of scarce biologically or medically useful products could have far-reaching benefits.

Cloned seventeen-nucleotide-long synthetic lactose operator is biologically active

A 17-nucleotide-long synthetic DNA molecule constituting the minimal recognition sequence of the lactose operator has been cloned in E. coli using the vehicle pBR313 and a synthetic HindIII adaptor. The clones containing the lac-pBR313 hybrid DNA constitutively produced beta-galactosidase. The level of beta-galactosidase was high and comparable to that obtained in cells carrying a 21-nucleotide-long synthetic lac operator on pMB9 plasmid or cells carrying a natural lac operator on pOP203-1 plasmid.

Studies on the lactose operon. III. Visualization and physical mapping of the lactose repressor-operator complex.

A physical mapping of the location of the lactose operator in the hybrid phage γh80dlacUV5 was accomplished by electron microscopy after mounting the lac repressor-lac DNA complex to a cytochrome c film followed by high-resolution shadowing. The location of the lac repressor peaked within a 0·15 μm wide zone at 3·2 μm from one end of the 15·2 μm long DNA molecule. The lac gene is thus located at 21% from one end of the DNA length.

Lactose Operator–Repressor Interaction

Publisher Summary This chapter discusses the lactose operator–repressor interaction. Any method for quantitatively studying the operator–repressor interaction requires either the operator or the repressor species to be labeled. After the interaction has taken place, the method must be able to separate the unreacted labeled material from the bound, labeled material. Two different methods have been used to study the operator–repressor interaction. Gilbert and Muller-Hill used 35 S-labeled lac repressor. The operator–repressor complex was separated from the unreacted lac repressor by zonal centrifugation on a 5–30% glycerol gradient. The method requiring labeled operator DNA is the one most commonly used. A convenient and accurate method has been developed and extensively used by Riggs and Bourgeois for studying various properties of the operator–repressor complex. In this method, the DNA–protein complex is separated from the unbound labeled DNA by passing the reaction mixture through a nitrocellulose membrane filter. The DNA–protein complex is retained on the filter, whereas the unbound DNA passes through. The chapter also discusses the lac operator interaction with a wild-type repressor.

[78] Lactose operator-Repressor interaction: Use of synthetic oligonucleotides in determining the minimal recognition sequence of the lactose operator

Publisher Summary This chapter discusses the use of synthetic oligonucleotides in determining the minimal recognition sequence of the lactose operator and studies the lactose operator-repressor interaction. The sequence of the lactose operator contains a 21-nucleotide-long region of twofold rotational symmetry. A number of O c mutants have been sequenced. All the mutations reside within a stretch of DNA 13-nucleotides long. Thus, the sequence of the lactose operator required for specific recognition by the lac repressor appears to be between 13 and 21 nucleotides in length. By synthesizing the various sequences in this region of lac operator DNA and then studying the binding properties of these synthetic molecules with the lac repressor, it is possible to determine the minimal recognition sequence.

Use of fluorene-9-methanol as a phosphate protecting group in the synthesis of deoxyribo-oligonucleotides

The fluore-9-methanol esters of the 5′-phosphates of oligonucleotides are soluble in organic solvents, and the protected oligonucleotides can be isolated by solvent extraction from the reaction mixture.