We developed a simple, rapid and reliable method to delete DNA fragments in plasmids using a polymerase chain reaction based amplification of the circular DNA sequence that excludes the fragment to be deleted. The primers are designed to contain a non-complementary 5' sequence consisting of a restriction enzyme target sequence. Following PCR amplification, the plasmid is digested with Dpn I to ...

Background: Indirect genetic diagnosis using polymorphic DNA markers can detect carriers of hemophilia A. This technique is preferable in developing countries because of its simplicity and cost effectiveness compared to direct mutation analysis. In the present study, we examined usefulness of intragenic marker BclI restriction fragment length polymorphism (RFLP) at intron 18, for carrier detect...

Type I restriction endonucleases are intriguing, multifunctional complexes that restrict DNA randomly, at sites distant from the target sequence. Restriction at distant sites is facilitated by ATP hydrolysis-dependent, translocation of double-stranded DNA towards the stationary enzyme bound at the recognition sequence. Following restriction, the enzymes are thought to remain associated with the...

Most well-known restriction endonucleases recognize palindromic DNA sequences and are classified as Type IIP. Due to the recognition and cleavage symmetry, Type IIP enzymes are usually found to act as homodimers in forming 2-fold symmetric enzyme-DNA complexes. Here we report an asymmetric complex of the Type IIP restriction enzyme MspI in complex with its cognate recognition sequence. Unlike a...

Reports on the prevalence of abortion due to Chlamydia psittaci (Chlamydophila abortus) in Kerala have indicated a need for the in depth study of the organism. To unearth differences among the isolates of Chlamydia psittaci, at molecular level, restriction enzyme analysis of genomic DNA and plasmid profiling were carried out. Four isolates from four different sources were used in this study. DN...

To generate DNA deletions, a tandem array of class IIS restriction enzyme recognition sites was cloned into a plasmid. The recognition sites were arranged so that each enzyme cleaves at a different site within an adjacent target sequence. Digestion with both enzymes followed by end repair and ligation resulted in the deletion of DNA between the two sites of cleavage. Because both recognition si...

We have found that, in the presence of a thermophilic restriction endonuclease, thermophilic DNA polymerase efficiently synthesizes and amplifies DNA in the absence of any added template and primer nucleic acid under isothermal conditions. More than 10 microg of DNA can be synthesized by 1 unit of DNA polymerase in 1 h, and the reaction proceeds until available dNTPs are consumed. We used mostl...

A sensitive assay for O6-alkylguanine-DNA alkyltransferase activity in cell or tumor extracts has been devised. The theoretical basis of the new assay lies in the observation that certain restriction enzymes will not cleave DNA containing methylated bases. Thus, if a synthetic oligodeoxynucleotide with a restriction sequence containing O6-methylguanine were incubated with the restriction enzyme...

The families of TGF-β proteins are the most important growth factors in the ovary for growth and differentiation of early ovarian follicles. Three related oocyte-derived members of the transforming growth factor-β superfamily namely growth differentiation factor 9 (GDF9), BMP15 and BMPR-IB have been shown to be essential for follicular growth and ovulation. Different mutations in the GDF9 gene ...

Nicotiana tabacum chloroplast DNA contains two copies each of 16S and 23S rRNA genes. These genes are located in an inverted order as determined from restriction fragment mapping and Southern hybridization to restriction fragments. The position of these genes on the N. tabacum chloroplast DNA molecule has been established relative to a complete map of SalI and SMaI restriction enzyme cleavage s...