Exponential Megapriming PCR (EMP) Cloning-Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.National Institutes of Health (U.S.) (Grant GM077537

Rapid Assembly of Multiple-Exon cDNA Directly from Genomic DNA

Backgrouud. 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 steps: harvest of tissues that express the gene of interest, RNA isolation, cDNA synthesis (reverse transcription), and PCR amplification. To simplify the cloning procedures and avoid the problems caused by ubiquitously distributed durable RNases, we have developed a novel strategy allowing the cloning of any cDNA or open reading frame (ORF) with wild type sequence in any spliced form from a single genomic DNA preparation. Methodology. Our Genomic DNA Splicing technique contains the following steps: first, all exons of the gene are amplified from a genomic DNA preparation, using software-optimized, highly efficient primers residing in flanking introns. Next, the tissue-specific exon sequences are assembled into one full-length sequence by overlapping PCR with deliberately designed primers located at the splicing sites. Finally, software-optimized outmost primers are exploited for efficient amplification of the assembled full-length products. Conclusions. The Genomic DNA Splicing protocol avoids RNA preparation and reverse transcription steps, and the entire assembly process can be finished within hours, Since genamic DNA is more stable than RNA, it may be a more practical cloning strategy for many genes, especially the ones that are very large and difficult to generate a full length cDNA using oligo-dT primed reverse transcription. With this technique, we successfully doned the full-length wild type coding sequence of human polymeric immunoglobulin receptor, which is 2295 bp in length and composed of 10 exons. © 2007 An et al.published_or_final_versio

DNA Ligase C and Prim-PolC participate in base excision repair in mycobacteria

Prokaryotic Ligase D is a conserved DNA repair apparatus processing DNA double-strand breaks in stationary phase. An orthologous Ligase C (LigC) complex also co-exists in many bacterial species but its function is unknown. Here, we show that the LigC complex interacts with core BER enzymes in vivo and demonstrate that together these factors constitute an excision repair apparatus capable of repairing damaged bases and abasic sites. The polymerase component, which contains a conserved C-terminal structural loop, preferentially binds to and fills-in short gapped DNA intermediates with RNA and LigC ligates the resulting nicks to complete repair. Components of the LigC complex, like LigD, are expressed upon entry into stationary phase and cells lacking either of these pathways exhibit increased sensitivity to oxidising genotoxins. Together, these findings establish that the LigC complex is directly involved in an excision repair pathway(s) that repairs DNA damage with ribonucleotides during stationary phase

Construction and analysis of recombinant DNA for human chorionic somatomammotropin

DNA complementary to mRNA coding for the human polypeptide hormone, chorionic somatomammotropin, has been purified by specific restriction endonuclease digestion and religation before cloning into bacterial plasmids. The primary structure of a major portion of this mRNA species is deduced from the nucleotide sequence of the recombinant DNA