BACKGROUND Polymerase chain reaction (PCR) is extensively applied in gene cloning. But due to the existence of introns, low copy number of particular genes and high complexity of the eukaryotic genome, it is usually impossible to amplify and clone a gene as a full-length sequence directly from the genome by ordinary PCR based techniques. Cloning of cDNA instead of genomic DNA involves multiple ...

efficiency was not affected by DNA source or PCR condition. Blue colonies contained a blunt-end-ligated plasmid without insert. Twenty randomly chosen white colonies were sequenced. Nineteen of them contained inserts ended with a primer and an A/T pair formed by complementary overhangs, and one contained an abnormally formed blank plasmid that was missing a part of the original sequence. The nu...

Direct cloning of PCR fragments by TA cloning or blunt end ligation are two simple methods which would greatly benefit high-throughput (HTP) cloning constructions if the efficiency can be improved. In this study, we have developed a ribosomal binding site (RBS) switching strategy for direct cloning of PCR fragments. RBS is an A/G rich region upstream of the translational start codon and is esse...

For most purposes, cloning protocols based on the polymerase chain reaction (PCR) using either the template-independent terminal transferase activity of Taq DNA polymerase in TA cloning schemes (8,9) or blunt-endbased procedures involving Pwo or Pfu DNA polymerase or polished Taq-generated PCR products are quite satisfactory. Nevertheless, both techniques have been proven less straightforward t...

MATERIALS Bacteriophage T4 DNA ligase T vector Target DNA (25 μg/ml), amplified by PCR When the PCR mixture contains more than one or two bands of amplified DNA, purify the target fragment by electrophoresis through low melting/gelling temperature agarose (please see Recovery of DNA from Low-meltingtemperature Agarose Gels: Organic Extraction). If not purified by gel electrophoresis, PCR-amplif...

We have developed a novel coincidence cloning strategy, termed Coincidence Painting, which enables the rapid generation of large numbers of region specific sequences. Coincidence Painting utilises Degenerate Oligonucleotide Primed PCR (DOP-PCR) amplification of flow sorted derivative translocation chromosomes. The PCR products are hybridised in situ onto specific flow sorted chromosomes for coi...

Many different strategies are used for cloning polymerase chain reaction (PCR) products. Some use restriction sites pre-integrated into primers or contained in a generated fragment. Others, such as the one used by Stratagene (La Jolla, CA, USA) in its pCR-Script Direct SK(+) Cloning Kit, are based on a blunt-end ligation. These strategies require the use of additional enzymes to polish the end...

INTRODUCTION Incubation of a blunt-end ligation reaction in the presence of an excess amount of an appropriate restriction enzyme can dramatically increase the yield of recombinant plasmids. The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are re-formed when linear, blunt-ended plasmid molecules ligate to themselves. In almost all cases, ...

The random-amplified polymorphic DNA (RAPD) technique (5,7) is one of the most useful methods for species identification and studies on the genetic structure of populations of microand macro-parasites. This method generally provides numerous markers that must be cloned and labeled to be used as probes (1,4). These probes can be used to test the specificity and the polymorphism of the RAPD marke...