Sequencing Nucleic Acids

In this chapter, the Maxam-Gilbert method and the Sanger method for ab initio sequencing of long nucleic acid chains are discussed. Two alternative methods for sequencing of shorter DNA strands are described in chapter 5 with DNA arrays, sequences of up to a few hundred base pairs can be determined (section 5.3), while pyrosequencing is capable of identifying sequences of up to 50 bp (section 5.4). 

After fragmenting the DNA molecules, denaturing the fragments into ssDNA and separating these ssDNA via electrophoresis, sequencing of the bases within the fragments can be commenced. 

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Williams J G K 1997 Single-molecule detection of specific nucleic acid sequences in unamplified genomic DNA Anal. Chem. 69 3915-20... 

EMBL (European Molecular Biology Laboratory) [33] is a nucleotide sequence database provided from the online host EBl. Release 73 (December, 2002) consists of over 20 million nucleotide sequences with more than 28 billion nucleotides. The information includes sequence name, species, sequence length, promoter, taxonomy, and nucleic acid sequence. 

GenBank National Center for Biotechnology Information, USA nucleic acid sequence biblio., sub- stance, se- quence 22 mio sequences 28 billion bases journals, author submis- sions STN online daily www.ncbi.nlm.- nih.gov... 

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... 

GENESEQ InteUiGenetics, STN Derwent Information Ltd. international, biotechnology limited bibhographic data polypeptide and nucleic acid sequences and related indexing... 

MO Dayhoff, WC Barker, PJ McLaughlin. Inferences from protein and nucleic acid sequences Early molecular evolution, divergence of kingdoms and rates of change. Orig Life 5 311-330, 1974. 

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. 

Write the complementary nucleic acid sequence that would pair with each of the following DNA sequences ... 

The amino acid sequence of our first aPNA (which we termed backbone 1 or bl) was designed based on this amphipathic hehx sequence (Fig. 5.3 B). Specifically, this aPNA backbone included hydrophobic amino acids (Ala and Aib), internal salt bridges (Glu-(aa)3-Lys-(aa)3-Glu), a macrodipole (Asp-(aa)i5-Lys), and an N-ace-tyl cap to favor a-helix formation. The C-termini of these aPNA modules end in a carboxamide function to preclude any potential intramolecular end effects. Each aPNA module incorporates five nucleobases for Watson-Crick base pairing to a target nucleic acid sequence. 

The following is a list of Web sites that teadets may find useful. The sites have been visited at various times by one of the authots (RKM). Most ate located in the USA, but many provide extensive finks to international sites and to databases (eg, for protein and nucleic acid sequences) and onhne journals. RKM would be grateful if readers who find other useful sites would notify him of their URLs by e-mail (rmurray6745 rogers. com) so that they may be considered for inclusion in fumre editions of this text. 

Some of their derivatives have been used as antiviral drugs. Due to their flexible chemistry, they can be exploited to design drug delivery systems and in molecular nanotechnology. In such systems, they can act as a central lipophilic core and different parts like targeting segments, linkers, spacers, or therapeutic agents can be attached to the said central nucleus. Their central core can be functionalized by peptidic and nucleic acid sequences and also by numerous important biomolecules. 

Lamhoujeb, S., Fliss, I., Ngazoa, S. E., and Jean, J. (2008). Evaluation of the persistence of infectious human noroviruses on food surfaces by using real-time nucleic acid sequence-based amplification. Appl. Environ. Microbiol. 74, 3349-3355. 

BRANCHED DNA SIGNAL AMPLIFICATION FOR DIRECT QUANTITATION OF NUCLEIC ACID SEQUENCES IN CLINICAL SPECIMENS... 

Detecting the presence of a specific nucleic acid sequence may be sufficient for some applications, but there is an increasing demand for quantitation of the... 

The molecular sensitivities of the first and second generations of the bDNA assays were limited by nonspecific hybridization between the amplification probes and other nucleic acids. Short regions of hybridization between any of the probes constituting the amplification system, (preamplifier, amplifier, and labeled probe) and any nontarget nucleic acid sequence leads to amplification of the background signal. Capture probes, capture extenders, and sample nucleic acid are all sources of this background hybridization (Collins et al 1997). 

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... 

In bDNA the number of target molecules is not altered. The signal of direct hybridization rather than the nucleic acid sequence itself is amplified and thus is directly proportional to the amount of target sequence present in the clinical sample. Both RNA and DNA sequences can be measured directly in clinical specimens, and there is no need to transcribe RNA into cDNA as there is with PCR. 

In summary, bDNA has a number of distinct theoretical and practical advantages over target amplification systems for direct quantitation of specific nucleic acid sequences. The following sections review the specific clinical and research applications of this technology. 

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). 

Compton, J. (1991). Nucleic acid sequence-based amplification. Nature 350,91-92. 

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