Nucleic acid sequencing method

Staden, R. (1999). Finding protein coding regions in genomic sequences. In Doolittle, R. (ed.), Molecular Evolution Computer Analysis of Protein and Nucleic Acid Sequences, Methods in Enzymology. vol. 183. Academic Press, San Diego. 

Blackwell TK (1995) Selecrion of protein binding sites from random nucleic acid sequences. Methods Enzymol 254 604-618... 

Hoser, M. J. Nucleic acid sequencing methods, kits and reagents. PCT Int. Appl. WO 2004074503, 2004. 

Sanger was a corecipient of a second Nobel Prize in 1980 for devising methods for sequencing nucleic acids Sanger s strategy for nucleic acid sequencing will be described in Section 28 14... 

This chapter describes the chemistry of nucleotides and the m or classes of nucleic acids. Chapter 12 presents methods for determination of nucleic acid primary structure (nucleic acid sequencing) and describes the higher orders of nucleic acid structure. Chapter 13 introduces the molecular biology of recombinant DNA the construction and uses of novel DNA molecules assembled by combining segments from other DNA molecules. 

The quantitation of HCV RNA in serum may be important in predicting and monitoring response to antiviral therapy (Davis, 1994). A variety of methods have been used to quantitate HCV RNA, including endpoint dilution RT-PCR, competitive PCR, multicyclic RT-PCR, nucleic acid sequence-based amplification, RT-PCR with a single internal quantitation standard, and bDNA (Chazouilleres et al, 1994 Detmer et al., 1996 Ishiyama et al, 1992 Kaneko et al., 1992 Klevits et al., 1991 Kobayashi etal., 1993 Miskovsky etal., 1996). 

Nucleic acid hybridization methods use oligonucleotide DNA probes with sequences complementary to a portion of the nucleic acid of the target bacterium38,60 and designed to hybridize with immobilized DNA or RNA on a membrane. After any unbound probe has been washed off, the hybridized probe can be detected.64 66... 

The viral load test quantifies viremia by measuring the amount of viral RNA. There are several methods used for determining the amount of HIV RNA reverse transcriptase-coupled polymerase chain reaction, branched DNA, and nucleic acid sequence-based assay. Each assay has its own lower limit of sensitivity, and results can vary from one assay method to the other therefore, it is recommended that the same assay method be used consistently within patients. 

The last section of this chapter includes in brief a procedure of McF adden (9) for in situ hybridization. In situ hybridization relies on the complementarity of the bases contained within DNA and RNA. In addition to the hybridization (reassociation) of complementary DNA strands, hybridization is possible between DNA and RNA strands that are complementary. Also, hybridization is possible between a synthetic sequence and a sequence of biological origin. In situ hybridization may be used to determine the location of a specific nucleic acid sequence within a cell. The procedure requires the use of a probe for the sequence of interest. The probe, in turn, must be complementary to the sequence of interest. The probe may be either single stranded or double stranded, DNA or RNA. There must exist a method by which to detect the probe. 

It is only natural that, to date, bioinformatics tools contribute most to the analysis of amino acid sequences. Only a small amount of current sequence data is subjected to direct experimentation. The majority of amino acid sequences currently accessible in public databases have been derived by in silico translations of nucleic acid sequence data, despite the fact that amino acid sequencing was introduced historically long before nucleic acid sequencing. It is hard to predict the future of the experimental generation of primary data. Certainly, sequencing of nucleic acids continues to become cheaper and faster, and novel techniques may further enhance the production of data. DNA chips are already used to detect differences between very similar sequences other methods may generate DNA data even more efficiently. 

In situ polymerase chain reaction (PCR) is a very powerful tool, which enhances our ahility to detect minute quantities of a rare, single copy number, target nucleic acid sequences in freshly frozen or paraffin-embedded intact cells or tissue sections (1-10). In 1986, the introduction of PCR methods opened new horizons and revolutionized research in all areas of molecular biology (11,12). Dr. Hasse and his coworkers in 1990 used multiple primers and successfully amplified the target nucleic acid sequences in intact cells by combining a traditional in situ hybridization protocol with a powerful PCR technology (13). 

Currently, there is a need for high-throughput determination of nucleic acid sequences. At present, detection systems most commonly employ fluorescence-based methods. However, wide spread applications of such methods are limited by low speed, high cost, size, and number of incubations steps, among other factors. Application of electrochemical methods in affinity DNA sensors presents likely a promising alternative, allowing miniaturization and cost reduction, and potentially allowing application in point-of-care assays. 

A very readable text describing methods used to analyze protein and nucleic acid sequences. Chapter 5 provides one of the best available descriptions of how evolutionary trees are constructed from sequence data. 

Detection of low-abundance nucleic acid sequences For example, viruses that have a long latency period, such as HIV, are difficult to detect at the early stage of infection using conventional methods. PCR offers a rapid and sensitive method for detecting viral DNA sequences even when only a small proportion of cells is harboring the virus. 

This method provided a simple and fast procedure to identify the presence of a nucleic acid sequence with good sensitivity. On average, the liposomes entrapped several thousand dye molecules, which provided intrinsic amplification of individual binding events. A qualitative measure of nucleic acid hybridization by visualization of the colour on the dip stick was intrinsically provided and quantitative measurements were also possible by use of a reflectometer. This approach resulted in a limit of detection of 1 fmol and a dynamic range of over two orders of magnitude. Maximum signal-to-noise was obtained with 0.2 mol% of probe sequence immobilized on the liposomes. 

The methods of nucleic acid sequencing have now advanced to such an extent that the United States and other national governments are funding a 10-20 year project to sequence the entire human genome [10,11] at an estimated cost of 3 billion. The genome is the complete genetic blueprint of a human being. It consists of twenty four distinct chromosomes (twenty two pairs of autosomes... 

Doolittle, R.F. 1990. Molecular evolution Computer analysis of protein and nucleic acid sequences. In Methods in Enzymology, Vol. 183, R. F. Doolittle, Ed. Academic Press, Inc. San Diego, Chapter 6. 

The application of in situ hybridization (ISH) has advanced from short lived, non-specific isotopic methods, to very specific, long lived, multiple color fluorescent-ISH probe assays (FISH). Improvements in the optics, filter technology, microscopes, cameras, and data handling by software, have allowed for a cost effective FISH setup to be within reach of most researchers. The application of mFISH (multiplex-FISH), coupled to the advances in digital imaging microscopy, have vastly improved the capabilities for non-isotopic detection and analysis of multiple nucleic acid sequences in chromosomes and genes (1). 

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