Nucleic acid purification

Classical gel electrophoresis has been used extensively for protein and nucleic acid purification and characterization [9, 10], but has not been used routinely for small molecule separations, other than for polypeptides. A comparison between TLC and electrophoresis reveals that while detection is usually accomplished off-line in both electrophoretic and TLC methods, the analyte remains localized in the TLC bed and the mobile phase is immediately removed subsequent to chromatographic development. In contrast, in gel electrophoresis, the gel matrix serves primarily as an anti-... 

This company founded in 1994, is a contract molecular biology and screening service company for the pharmaceutical, research, and clinical markets worldwide. Custom services include gene synthesis, DNA sequencing, site-directed mutagenesis, nucleic acid purification, and genotype/genetic analysis. 

A prerequisite step to any rDNA work is the initial isolation of DNA or RNA from the source material (which can be microbial, plant, animal or viral). Numerous methodologies have been developed to achieve nucleic acid purification, and some of these methodologies have been adapted for use in a variety of commercially available purification kits. Although details vary, the general... 

Nucleic Acid Purification after Reverse Transcription... 

The degree of automations has literally revolutionized nucleic acid purification. The core of these systems are the automated liquid-handling workstations that involve the movement of multiple probes in Cartesian axes (x, y, z) over a deck configured with lab ware, such as microplates, tube racks, solvent reservoirs, washbowls, and disposable tips. They have the ability of aspirating and dispensing solvents from a source to a destination. Devices such as vacuum manifolds, heating blocks, and shakers have also been modified to handle 96-well (or more) plates. The multiprobe liquid handlers have a variable tip spacing that allows them to expand their tip-to-... 

Affinity chromatography Has a wide number of uses and can be applied to the isolation and purification of virtually all biomolecules. Specific applications include nucleic acid purification, protein purification from cell and tissues extracts, and antibody purification from blood serum. There are a number of matrices used for the construct, and some examples of these and their uses are as follows heparin columns to separate cholesterol lipoproteins, lectin columns to separate carbohydrate groups, and phenyl boronate columns to separate glycated haemoglobins. 

PCR is a resilient process and does not require highly purified nucleic acid. In practice, however, clinical samples may contain unpredictable amounts of impurities that can inhibit polymerase activity. To ensure reliable amplification, some form of nucleic acid purification is often used. The idiosyncratic nature of PCR inhibitors within clinical specimens requires demonstration that the sample (or preparation of nucleic acid purified from it) will allow amplification. A control nucleic acid sequence, usually different from the target, can be added to the sample (or extract from the sample). Failure to amplify this control indicates that further purification of the sample is required to remove inhibitors of the reaction. 

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. 

Cady NC, SteUck S, Batt CA (2003) Nucleic acid purification using microfabricated sdicon structures. Biosens Bioelectron 19 59-66... 

Many of the techniques used in protein purification procedures have also been adapted for use with nucleic acids. For example, several types of chromatography (e.g., ion-exchange, gel filtration, and affinity) have been used in several stages of nucleic acid purification and in the isolation of individual nucleic acid sequences. Because of its speed, HPLC has replaced many slower chromatographic separation techniques when small samples are involved. 

Sample extraction involves purifying a single component from a complex mixture. It usually is based on exploiting a unique property of the component of interest such as solubility or charge which allows it to be separated from other components in solution. Sample extraction is often referred to in the context of nucleic acid purification from a complex biological mixture such as a cell lysate. 

Quake and coworkers [16] developed a PDMS microfluidic device (shown in Fig. 4c) for nucleic acid purification from a small number of bacterial or mammalian cells. This multilayer device contained fluidic channels and a system of membrane-actuated pneumatic valves and pumps, which enabled precise control of buffers, lysis agents, and cell solution and also allowed for parallel processing. Bacterial cells, dilution buffer, and lysis buffer are first introduced into the chip and then transferred into the rotary mixer. Once mixed, the lysate is flushed over a DNA affinity column and drained. The DNA... 

Finally, the transcripts were completely digested by RNase Ti at 37°C for 10 min with MALDI matrix (3-HPA) added as a denaturant. Briefly, 5 pL of RNA transcript (up to 20 pg) is added to 4 pL 3-HPA in 50% ACN/water and 1 pL of RNase Tj (1000 units) and reacted for 10 min and then placed on ice for MALDI preparation. For highest quality spectra, samples are desalted by reverse-phase purification in ZipTips according to manufacturer s directions for nucleic acid purification [96]. The final step in this process is elution of purified RNA oligonucleotide fragments onto the MALDI target using 2 pL of the MALDI matrix itself. Samples (MALDI spots ) are allowed to air dry or may be rapidly dried under vacuum. 

Affinity chromatography can be used in a number of applications, including nucleic acid purification, protein purification from cell free extracts, and purification from blood. 

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