Nucleic acid sequencing transcription

Both target and signal amplification systems have been successfully employed to detect and quantitate specific nucleic acid sequences in clinical specimens. Polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR) are all examples of enzyme-mediated, target amplification strategies that are capable of producing billions of... 

Molecular biology involves the study of the major macromolecules, DNA, RNA, and protein. The central dogma ofmolecular biology is illustrated in Fig. 2. The central dogma shows the relationship among the macromolecules in the processes of transcription and translation. Figure 2 also gives the relationship between immunoelectron microscopy and in situ hybridization. In situ hybridization allows one to localize a specific nucleic acid sequence. Immunoelectron microscopy is an essential component to the technique of in situ hybridization when applied at the EM level. 

More detailed RT-PCR analyses of mRNA from a variety of tissues have revealed additional structural complexity and identification of a large number of alternatively spliced subunit transcripts that differ in variable domain nucleic acid sequence (Tombes et al. 2003). These subunit variants are expressed in a tissue-specific manner. For example, the 6i subunit mRNA is expressed predominantly in brain, whereas S2 and 63 variants are expressed in rat aortic smooth muscle,... 

Decoy methods production of synthetic decoys corresponding to a specific nucleic acid sequence which binds virally encoded regulatory proteins and affects transcription. 

Transcription-based amplification methods are modeled after the replication of retroviruses. These methods are known by various names including nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA)/ and self-sustained sequence repfi-cation (3SR) assays. Isothermal target amplification, using the collective activities of reverse transcriptase, RNase H, and RNA polymerase, is common to these methods. As illustrated in Figure 37-6, the method may be applied to... 

RT-PCR, Reverse transcription-polymerase chain reaction NASBA, nucleic acid sequence based amplification. For current FDA listings, see http //www.accessdata.fda.gov/scripts/cdrli/cfdocs/cERL/lLsting.cfiii. 

This flow of information depends on the genetic code, which defines the relation between the sequence of bases in DNA (or its mRNA transcript) and the sequence of amino acids in a protein. The code is nearly the same in all organisms a sequence of three bases, called a codon, specifies an amino acid. There is another step, between transcription and translation, in the expression of most eukaryotic genes, which are mosaics of nucleic acid sequences called introns and exons. Both are transcribed, but introns are cut out of newly synthesized RNA molecules, leaving mature RNA molecules with continuous exons. The existence of introns and exons has crucial Implications for the evolution of proteins. 

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... 

Nucleic acid sequence databases typically contain sequence data, which includes information at the level of the gene structures, introns and exons (for eukaryotics), cDNA (complementary DNA), RNA and transcription regulations. The important nucleic acid sequence data repositories as the primary resources known as International Nucleotide Sequence Database Collaboration (INSDC) are ... 

In addition to PCR, there are many other technologies to amplify nucleic acids. For example, ligation-based amplification or ligase chain reaction uses sequence-directed oligonucleotide primers and thermostable DNA ligase to assay point mutations, deletions, or insertions in DNA. Strand-displacement amplification uses the inherent strand-displacement activity of DNA polymerases to conduct DNA amplification at a constant temperature. Transcription-based methods such as nucleic acid sequence-based amplification (NASBA) involve in vitro RNA transcription. NASBA and most other transcription-based... 

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