DNA purification methods

The DNA extraction method is an in-house developed protocol. Microdissected tumor area are dipped into xylene to remove paraffin, rehydrated in a series of ethanol, and incubated in a proteinase K digestion buffer. Two different DNA purification methods are used. The first method is a NaCl saturated solution precipitation, as previously described. In the second method we use reagents and materials provided by the QIAamp DNA Blood... 

The advent of recombinant DNA technology led to the development of antibodies and fragments that are tailored for optimal behaviour in vivo [7,8]. Humanized and chimeric antibodies can be constructed to circumvent the human anti-mouse antibody response elicited by mouse antibody treatment of patients, which severely hampers the application of these powerful molecules. The treatment of rheumatoid arthritis patients with doses of as high as 10 mg kg cA2 chimeric antibody specific for TNFa [9], emphasizes that at present the production and purification methods for these proteins have been optimized to such extent that clinical studies can be considerably intensified. 

These priming methods therefore offer a powerful way of sequencing rare mRNA species that are present in a heterogeneous population of mRNAs without resort to either purification methods (e.g. immunoprecipitation) or cloning methods. The principal drawback is the length of sequence that can be determined from a single 5 -end labelled primer (about 250 nucleotides) and the fact that the data obtained relies on the sequencing of only one DNA strand. 

It is important to note that no single method of cell lysis or DNA purification will be appropriate for all environmental samples. Therefore, researchers should consult protocols that may apply to their systems of study in order to optimize DNA recovery from novel environmental samples. 

Instead of specific amplification of one target to improve sensitivity, methods that amplify all genomic DNA or mRNAs are useful when the target is in short supply. For example, multiple-displacement amplification uses exonuclease-resistant random hexamers and a highly pro-cessive polymerase to amplify DNA nonspecificaily. Initial DNA denaturation is not necessary and the reaction proceeds isothermally. Similarly, messenger RNA can be generi-caUy amplified with a poly(T) primer modified with an RNA polymerase promoter. After reverse transcription, second-strand DNA synthesis, and transcription, antisense RNA is produced. Both whole genome and antisense RNA amplification are also useful as nucleic acid purification methods before amplification or detection. 

Methods for detecting DNA from whole cells on nylon, without DNA purification and processing of the samples in individual vials (Brandsma and Miller, 1980), have recently been developed (McIntyre and Stark, 1989 Reed and Matthaei, 1990 Hammermueller et al., 1991). These procedures avoid enzymatic dispersion of cells, RNase and pronase treatments to hydrolyze cellular macromolecules, etc. These alternative methods are based on the capacity of hot alkali to disperse and solubilize cells and hydrolyze macromolecules including RNA and protein, but not DNA. Positively charged modified nylon membranes then irreversibly bind nucleic acid (Reed and Mann, 1985) while remaining suitable for hybridization. Critical parameters in this procedure are the temperature (80°C), the length of the incubation period (20 min) and the NaOH... 

Plasmid is routinely recovered from bacteria by an alkaline lysis procedure, which lyses the bacterial cell while maintaining bacterial DNA attachment to the cell wall. This procedure enables subsequent precipitation of bacterial DNA and cellular debris, leaving a crude preparation enriched in plasmid. We routinely use a plasmid DNA purification kit provided by Qiagen that utilizes the alkaline lysis method for harvesting, and anion exchange column chromatography for rapid purification. We refer the reader to the detailed instructions provided in the kit by the manufacturer, which we have not found necessary to modify for purification of laboratory-use plasmid DNA. 

Plasmid purification methods vary in the time, expense, and equipment required and in the purity of the plasmid produced. Purity is important to generate high levels of transfection in a reproducible manner. Potential contaminants include bacterial genomic DNA, RNA, protein, endotoxin, chemical residues, trace metals, and undesirable counterions (159). Depending on the user s endpoint, concern regarding any of these contaminants will vary. For example, if the plasmid product is intended for human clinical trials, strict quality control over of all these factors, among others, must be addressed. Lesser concern is warranted for... 

Figure 10.9 Non-PCR random mutagenesis. Overview of a random site-directed mutagenesis method. Obtain template DNA [ssDNA (—) strand] Step 1 anneal spiked oligodeoxy nucleotide mutagenic primer (blue) with desired mutation range Step 2 extend and incorporate mutagenic primer into new (-f) strand containing dCTPaS Step 3 digest the template DNA (—) strand with Nci I and ExoIII, then reynthesize (—) strand to include mutations into (—) strand as well Step 4 transform E. coli with random mutant recombinant DNA plasmid ready for selection, DNA purification and sequence identification of mutant recombinant DNAs.

Figure 8.3 shows the process flow sheet for large-scale purification of plasmid DNA. Table 8.14 presents a comparison of laboratory methods and laige-scale pharmaceutical processes for plasmid DNA purification. 

M. Muller, Considerations for the scale-up of plasmid DNA purification in Nucleic Acid Isolation Methods Eds. B. Bowlen, P. Dtirre), American Scientific Publishers, New York, 2003. 

DNA template amounts from 5 to 50 ng from a variety of purification methods have been successfully used in this Fragment Size Analysis protocol. Of primary importance is the uniformity of the concentrations across all samples. This will aid in the uniformity of sample injection, providing higher quality data for downstream analysis. 

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