One of the common methods for cloning polymerase chain reaction (PCR) products is overhanging-end cloning (also known as sticky-end or directional cloning). Frequently, it is not possible to use restriction enzyme sites already present in the amplified product, and primers that encode recognition sites of restriction endonucleases in addition to the specific sequence have to be designed. After ...

BACKGROUND While conventional cloning methods using restriction enzymes and polynucleotide ligase are adequate for most DNAs, fragments made by the polymerase chain reaction are difficult to clone because the amplifying DNA polymerase tends to add untemplated nucleotides to the 3'-termini of the amplified strands. Conservative site-specific recombinases offer an efficient alternative to convent...

In vivo recombinational cloning in yeast is a very efficient method. Until now, this method has been limited to experiments with yeast vectors because most animal, insect, and bacterial vectors lack yeast replication origins. We developed a new system to apply yeast-based in vivo cloning to vectors lacking yeast replication origins. Many cloning vectors are derived from the plasmid pBR322 and h...

The cloning of polymerase chain reaction (PCR) products provides one with a stable form of the amplified segment and facilitates further manipulation and study of the target molecule. A number of cloning protocols have been developed either based on a restriction endonuclease recognition site built into the primers or by using the template-independent terminal transferase activity (“extendase” ...

Construction and screening of DNA libraries for genes of interest represent the standard but tedious cloning procedures (1). The advent of PCR technology has greatly enhanced the efficiency of traditional gene isolation and cloning techniques. Various strategies have been developed for PCR amplification of an unknown DNA fragment that flanks one end of a known sequence which include inverse PCR...

Antimicrobial activity of certain species Lactobacillus bacteria were known to produce a type bacteriocin called Plantaricin (Pln). In this study, the PlnF (plnF) gene (666 bp) encoding plantaricin was synthetically constructed based on sequence predefined and valuable amino acid compositions subcloned into pET-28 (+) expression vector. Thereafter transformation recombinant vector BL21 (DE3) co...

The polymerase chain reaction (PCR) has been extensively used for the mutation of DNA sequences (1–5,7,8). Many variations of the protocol have been implemented, but most involve a first step in which PCR is used to modify the molecule, and a second step in which the new sequence is subcloned into a vector. A new method, named inverse PCR mutagenesis (IPCRM) has been shown to be an efficient an...

background: as a drug target and an antigenic agent, hiv-1 protease (hiv-1 pr) is at the center of attention for designing anti-aids inhibitors and diagnostic tests. in previous studies, the production of the recombinant protease has been faced with several difficulties; therefore, the aims of this study were the easy production, purification of the soluble form of protease in e. coli and inves...