Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on...

We describe a simple method for the cloning of PCR products without the need for post-amplification enzymatic treatment. Tailed PCR primer sets are used to create complementary staggered overhangs on both insert and vector by a post-PCR denaturation-hybridisation reaction. The single-stranded overhangs are designed to allow directional cloning in a ligase-free manner. This 'enzyme-free cloning'...

TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase. Taq polymerase has non-template dependent activity which preferentially adds a single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most...

introduction: beta thalassemias are a heterogenous group of autosomal recessive disorders, characterized by reduced or absent production of the b-globin chain by the affected allele. transplantation of allogenic hematopoietic stem cells (hsc) is a curative approach but this therapeutic option is not available to the majority of patients. transplantation of genetically corrected autologous hsc i...

Cloning of polymerase chain reaction (PCR) products can be a valuable research technique, but in practice the technical problems associated with the methodology may limit its usefulness. A variety of methods have been developed that facilitate PCR product cloning. These include blunt-end ligation cloning (5), ligation-independent cloning (1,6), introduction of restriction sites into PCR primers...

By incorporating dUMP residues into the 5' end of PCR primers, one can generate products which, after treatment with uracil DNA glycosylase (UDG), contain 3' overhangs. These overhangs can be annealed to vector molecules with complementary overhangs generated in a similar fashion and transformed directly into Escherichia coli without the need for ligase. We have tested this method of ligation-i...

objectives: in present study we aimed to clone the lux i gene encoding n-acyl-homoserine synthase detected in biofilm forming clinical isolates of acinetobacter baumannii and study its expression in escherichia coli transformants. materials and methods: four a. baumannii hospital strains which demonstrated strong biofilm activity were selected in this investigation. the presence of lux i gene w...

Molecular cloning methods based on primer and overlap-extension PCR are widely used due to their simplicity, reliability, low cost and high efficiency. In this article, an efficient mega primer-mediated (MP) cloning strategy for chimaeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: (i) the ...

1.Atcheson, C.L., B. DiDomenico, S. Frackman, R.E. Esposito and R.T. Elder. 1987. Isolation, DNA sequence, and regulation of a meiosis-specific eukaryotic recombination gene. Proc. Natl. Acad. Sci. USA 84:80358039. 2.Cha, J., W. Bishai and S. Chandrasegaran. 1993. New vectors for direct cloning of PCR products [published erratum appears in Gene 1994; 141:149]. Gene 136:369-370. 3.Clark, J.M. 19...