Purification of genomic DNA with minimal contamination of proteins.

The purification is based on a set of solutions and a simple centrifugation procedure. Protocols are designed for an easy extraction and purification of genomic DNA from a wide range of samples, including whole blood, buffy coat, bone marrow, body fluids, buccal cells, tissues, mouse tails, etc. RBCs are lysed by dilution into a hypotonic solution. Tissues are broken down and digested by proteinase K in the presence of an anion detergent to release genomic DNA. After precipitation of the detergent and proteins, unique beads that bind proteins, lipids, and RNAs are added to achieve the supreme purity. Genomic DNA is then separated by alcohol precipitation. A proprietary nucleic acid precipitation reagent is used to enhance DNA recovery from low concentration samples. No DNA-binding beads or columns are used in the method, eliminating the problem of low yield and the risk of shearing of genomic DNA. The purified samples are free of proteins, lipids, salts, and RNA contamination. Purified samples are also stable for storage and suitable for all downstream applications.

A method for preparing DNA sequencing templates using a DNA-binding microplate.

A DNA-binding matrix was immobilized on the surface of a 96-well microplate and used for plasmid DNA preparation for DNA sequencing. The same DNA-binding plate was used for bacterial growth, cell lysis, DNA purification, and storage. In a single step using one buffer, bacterial cells were lysed by enzymes, and released DNA was captured on the plate simultaneously. After two wash steps, DNA was eluted and stored in the same plate. Inclusion of phosphates in the culture medium was found to enhance the yield of plasmid significantly. Purified DNA samples were used successfully in DNA sequencing with high consistency and reproducibility. Eleven vectors and nine libraries were tested using this method. In 10 microl sequencing reactions using 3 microl sample and 0.25 microl BigDye Terminator v3.1, the results from a 3730xl sequencer gave a success rate of 90-95% and read-lengths of 700 bases or more. The method is fully automatable and convenient for manual operation as well. It enables reproducible, high-throughput, rapid production of DNA with purity and yields sufficient for high-quality DNA sequencing at a substantially reduced cost.

High performance DNA purification using a novel ion exchange matrix.

Ion exchange chromatography has emerged as a reliable alternative to classic CsCl-ethidium bromide gradients for isolating nucleic acids of the highest purity. A plasmid purification method based on a unique anion exchange membrane (IEXM) was developed for the production of superior quality plasmids. This method was simpler and more efficient than conventional bead-based methods. Plasmids were extracted from bacterial cells through alkaline lysis. The crude lysate was clarified by a sequential filtration device that not only removed cell debris but micellar aggregates as well. The clarified lysate was mixed with an extraction solution and loaded into a spin column containing IEXM. Binding, washing, and elution conditions were optimized to achieve efficient isolation of plasmids from the impurities. IEXM had an exceedingly high dynamic binding capacity, excellent selectivity, and a near 100% recovery for plasmids. The binding capacity for pUC19 was 2.93 mg/cm(3) of IEXM, which is several times greater than the values for conventional ion exchange beads. The superior selectivity of the method was reflected in the extremely low levels of endotoxin, and thus it is well-suited for critical applications in eukaryotic systems.

Nkx2.1 transcription factor in lung cells and a transforming growth factor-beta1 heterozygous mouse model of lung carcinogenesis.

The Nkx2.1 homeobox gene and transforming growth factor-beta1 (TGF-beta1) are essential for organogenesis and differentiation of the mouse lung. NKX2.1 is a marker of human lung carcinomas, but it is not known whether this gene participates in early tumorigenesis. Addition of TGF-beta1 to TGF-beta1-responsive nontumorigenic mouse lung cells cotransfected with a NKX2.1Luc luciferase reporter and either a Sp1 or Sp3 plasmid showed a significant increase or decrease, respectively, in NKX2.1Luc transcription. Cotransfection of Sp3 and dominant-negative TGF-beta type II receptor plasmids negated the effect of Sp1. Cotransfected Sp1 plasmid with either dominant-negative Smad2 or Smad3 or Smad4 plasmids significantly decreased NKX2.1Luc transcription. Electrophoretic mobility shift assays revealed binding of Sp1 and Smad4 to the NKX2.1 promoter. With a TGF-beta1 heterozygous mouse model, Nkx2.1 mRNA and protein in lungs of TGF-beta1 heterozygous mice were significantly lower compared to wildtype (WT) littermates. Competitive reverse transcription (RT)-polymerase chain reaction (PCR) and immunostaining showed that Nkx2.1 mRNA and protein decreased significantly in adenomas and adenocarcinomas compared to normal lung tissue. Our in vitro data showed that regulation of Nkx2.1 by TGF-beta1 occurs through TGF-beta type II receptor and Smad signaling, with Sp1 and Sp3 in lung cells. Our in vivo data showed reduced Nkx2.1 in lungs of TGF-beta1 heterozygous mice compared to WT mice, that is detectable in adenomas, and that is further reduced in carcinogenesis, and that correlates with reduction of Sp1, Sp3, and Smads in lung adenocarcinomas. Our findings suggest that reduced Nkx2.1 and TGF-beta1 signaling components may contribute to tumorigenesis in the lungs of TGF-beta1 heterozygous mice.