Polymerase chain reaction nucleic acid separation

Biodistribution studies are conducted to evaluate the dissemination and persistence of nucleic acid/DNA and viral vector-based vaccines. Vector persistence should be examined at more than one time point up to at least 21 days or more after the last injection 22 unpublished data) and the biodistribution and persistence of a plasmid is usually dependent upon the route of administration.20 These studies can be combined with a general toxicology study or may be conducted separately. In most cases, the DNA is extracted from the selected tissues and assessed by quantitative Polymerase Chain Reaction (qPCR).1516... 

This chapter illustrates the characterizations of technical procedures necessary in the course of these molecular evaluations, describing our laboratory experience and suggesting different diagnostic approaches microdissection techniques, extraction of nucleic acids from paraffin-embedded tissues, polymerase chain reaction (PCR), direct sequencing, and allelic separation by cloning [4-7],... 

Nucleic acid analysis. Methods that analyse the microbial community from nucleic acid composition are based on use of the polymerase chain reaction (PCR). Universal forward and reverse primers are used in combination with PCR to amplify species-specific DNA fragments (usually the 16S subunit of ribosomal DNA) from samples isolated directly from soil. Samples then are separated... 

HPLC life science applications focus on the separation, quantitation, and purification of biomolecules such as proteins, peptides, amino acids, nucleic acids, nucleotides, and polymerase chain reaction (PCR) amplification products.31 34 These are diversified and active research areas in medical research and drug discovery. 

The process is initiated with a random library of linear oligonucleotides (usually, 10 to 10 ) consisting of linear nucleic acids comprising a random sequence embraced by a 5 and a 3 nucleic acid sequence of defined composition. An RNA-searched aptamer involves the primary transcription of the DNA library into an RNA pool followed by passing the library through a separating matrix that includes the target substrate. The few nucleic acids that reveal affinity toward the substrate (or some nonspecific nucleic acid adsorbents) bind to the separation matrix, while most of the library components are washed off. The elution of surface-bound nucleic acids followed by their polymerase chain reaction (PCR) amplification yields a mixture of nucleic acids... 

The melting temperature (T ) of nucleic acids defines the thermal point at which 50% of the complementary molecule dissociate/melt away from their cognate strands or anneal/hybridize the two strands. The capacity to estimate melting temperatures is important because it helps the researcher select strand separation/hybridization temperatures, such as in polymerase chain reaction and microarray assays. A good approximation of melting tonperature for oligonucleotides takes the expression (Wallance et al., 1979) ... 

The use of animal antibodies to detect antigens is highly effective but it involves the use of animals, complex separations, and has a variety of limitations. Starting around 1990 a new method for detection and separation of substances, including amino acids, proteins, and pharmaceuticals, was developed based on synthetic nucleic acids termed aptomers aptos = "to fit"). Aptomers are DNA or RNA molecules that bind with high specificity to certain molecules. Their application depends upon many principles described earlier in this book chromatography, combinatorial chemistry, and the polymerase chain reaction (PCR) technique. 

Extraction of DNA from biological samples can be accomplished by precipitation or affinity methods [15]. Amplification of the amount of DNA is also needed before any sequence detection can be done. This can be done by a method known as polymerase chain reaction or PCR in short. This process is depicted in Figure 8.6. Briefly, original double stranded DNA molecules, shown as black rectangles, are heated to more than 90°C for separation. Afterward, DNA primers, shown as squares, as well as nucleic acids are added to the solution to initiate the DNA synthesis, forming two pairs of double stranded DNA at the end of the first cycle. The process is repeated for more than 30 times, doubling the amount of DNA at each step [16]. RNA can also be amplified using a similar process known as reverse transcription-polymerase chain reaction (RT-PCR) [17]. 

As the name suggests, polymerase chain reaction (PCR) is a method of amplifying selected segments of target nucleic acid sequences (typically 150-3000 base pairs in length) by several orders of magnitude prior to separation and characterization. Since the pioneering research and... 

The success of genomics owes much to the polymerase chain reaction (PCR), the repeated use of strand separation, followed by synthesis of complementary double helices. Using PCR, tiny amounts of nucleic acid can be amplified and concentrated easily. There is no such universal amplification reaction for proteins, metabolites, sugars, and lipids. For this reason, it is quite likely that we know of only a tiny fraction of the proteins in existence, the concentration of most of them lying below current detection limits. 

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