DNA cleanup

Reagents required for the conventional method include sodium hydrogen sulfite (Sigma-Aldrich, cat. no. 243973), hydroquinone (Sigma), NaOH (3M), mineral oil. Wizard DNA Cleanup system (Promega), isopropanol, ammonium acetate (lOAf), glycogen, and ethyl alcohol. 

QIAquick PCR Purification Kit (Qiagen) The features of the kit are available on the Web site http //www.qiagen.com/Products/ Catalog/Sample-Technologies/DNA-Sample-Technologies/ DNA-Cleanup/QIAquick-PCR-Purification-Kit. 

Sample-Technologies/DNA-Sample-Technologies/DNA-Cleanup/DyeEx-20-Spin-Kit. 

Once the DNA has been isolated, it is important that any of the reagents used for cell lysis or inhibitor removal are completely washed away from the sample. DNA cleanup includes several washing steps to ensure that DNA is the only material present for subsequent experiments. 

The ability to detect small genetic changes becomes more difficult as mass increases. There is further an upper mass range where analysis is impractical. For low-resolution instruments this limit is around a 100 mer. Thus the mass has to be minimized or a high-resolution instrument employed. Alternatively, the smaller the piece of DNA analyzed, the more it chemically resembles a primer or nucleotide monomer thus separation of the two during cleanup is difficult to do. If the primers and nucleotides are not removed, they can provide a massive background on MS analysis or inhibit ionization of the PCR product by preferential ionization. Thus for practical reasons it is extremely difficult to employ a PCR product below a 40 to 50mer for direct ESI MS or ESI MS-MS analysis. 

We have found that cloning efficiency is reduced when increased volumes of purified PCR product are used, possibly as a result of carryover of contaminants from the cleanup reaction. Therefore, it is best to have the PCR product as concentrated as possible. The exact amount of DNA in this step can vary somewhat to allow for standardized pipeting volumes without compromising cloning efficiency. 

Smith and co-workers designed a microfabricated dialysis device for sample cleanup before ESI MS. A microdialysis membrane sandwiched between two chips having micromachined serpentine channels provided efficient desalting for both DNA and protein samples before subsequent ESI ion trap MS. In a continuation study, they used a fabricated dual microdialysis membrane for removing both high- and low-molecular-weight species that... 

After cleanup of the sequencing reaction either manually or in an automated system, the DNA fragments are separated on genetic analyzers. As older instruments... 

Several modifications of MALDI have been developed to couple additional sampling and reaction capabilities to this technique. Surface-enhanced laser desorption ionization (SELDI) is one type of modified MALDI and describes an ionization process that involves reacting a sample with an enhanced surface. With SELDI, the sample interacts with a surface modified with some chemical functionality prior to laser desorption ionization and mass analysis. For example, an analyte could bind with receptors or affinity media on the surface, and be selectively captured and sampled by laser desorption. A SELDI surface can be modified for chemical (hydrophobic, ionic, immunoaffinity) or biochemical (antibody, DNA, enzyme, receptor) interactions with the sample. This technique can act as another dimension of separation or sample cleanup for analytes in complex matrices. As discussed before, one disadvantage of MALDI is that the matrix (usually a substituted cinnamic acid) that is mixed with the sample can directly interfere with the analysis of small molecules. There have been several areas of research to overcome this issue.Direct ionization on silicon (DIOS) is an example of a modification of MADLI that eliminates the matrix. In this case, analytes are captured on a silicon surface prior to laser desorption and ionization. Other examples of matrix-free laser desorption techniques include the use of siloxane or carbon-based polymers. 

The systems described thus far addressed only a portion of the individual tasks required to elucidate DNA sequence. A more efficient process would fully integrate the tasks of DNA extraction, purification, template preparation, and amplification with sequencing reaction chemistry, post reaction cleanup, separation, and detection [152]. Several groups have explored partially integrated systems intended to streamline various steps in this process [152-156],... 

In 1998, Karger and coworkers [158,159] published a two-part study on the role of sample matrix components and post reaction sequencing product cleanup. In this study, the authors used ultra-filtration to remove template DNA and gel filtration to reduce salt concentration in the sample... 

Ruiz-Martinez, M.C., Salas-Solano, O., Carrilho, E., Kotler, L., and Karger, B.L., A sample purification method for ragged and high-performance DNA sequencing by capillary electrophoresis using replaceable polymer solutions. A. Development of the cleanup protocoL Anal. Chem., 70, 1516, 1998. 

There is great potential for integration of all stages of PCR-MS and PCR-MS/MS analysis including sample collection, DNA isolation, PCR, sample cleanup, MS/MS, and computer-assisted data handling. As noted above, with the exception of sample clean-up, the other stages are identical for real-time PCR and... 

---