Sequencing methods

Pentapeptide consisting of five amino acid residues (A-E) 

I Residual tripeptide Process continued until E is reached 

Peptide segments obtained by a second type of cleavage 

The overlap information allows the original protein sequence to be generated. In this example, the sequence D-E-F-G in the fourth peptide proves that the first and second peptides must have been joined originaliy at E,F and not A,H. 

Protein sequencing by creation of overlap peptides (mapping), 

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The second generator is an arithmetic sequence method that generates random number using the following mathematical operation ... 

Cuff IA and G J Barton 1999. Evaluation and Improvement of Multiple Sequence Methods for P Secondary Structure Prediction. Proteins Structure, Function and Genetics 34 508-519. 

DNA from a gene contains hundreds to thousands of nucleotide units for which the sequence is needed in order to interpret its code. Sequencing methods require only small amounts (5 (tg) of purified DNA, which can be produced by cloning. Automated sequencers are available that can daily sequence DNA containing hundreds of nucleotide units. 

The STEP procedure, described by Hendrick and Benner (1987), was developed from a research program on incident investigation methods. STEP is based on the mulHple events sequence method and is an investigative process which structures data collection, representation, and analysis. 

In contrast, RNA occurs in multiple copies and various forms (Table 11.2). Cells contain up to eight times as much RNA as DNA. RNA has a number of important biological functions, and on this basis, RNA molecules are categorized into several major types messenger RNA, ribosomal RNA, and transfer RNA. Eukaryotic cells contain an additional type, small nuclear RNA (snRNA). With these basic definitions in mind, let s now briefly consider the chemical and structural nature of DNA and the various RNAs. Chapter 12 elaborates on methods to determine the primary structure of nucleic acids by sequencing methods and discusses the secondary and tertiary structures of DNA and RNA. Part rV, Information Transfer, includes a detailed treatment of the dynamic role of nucleic acids in the molecular biology of the cell. 

Molecular weight or number of sequences Method of preparation Applied phys. or chem. methods... 

Specific sequencing methods (eg, for proteins and nucieic acids)... 

Antibody-based detection methods include immuno-cytochemistry, which gives qualitative data but has very good spatial resolution. Radioimmunoassays provide a quantitative measure of release or content. One of the major limitations of all antibody-based methods is the potential for cross-reactivity among the many peptides. For example, some of the most sensitive gastrin antisera also detect CCK, since the peptides share a common COOH-terminal tetrapeptide sequence. Methods for detection of the mRNAs encoding neuropeptides include Northern blots, which provide quantitative data and information on splice variants, but lack fine anatomical resolution. The more commonly used polymerase chain reaction, which can be quantitative but often is used in a more qualitative manner, provides great sensitivity. Alternatively, in situ hybridization preserves anatomical relationships and can be used to obtain both qualitative and quantitative data. 

The use of magnetic resonance imaging (MRI) to study flow patterns in reactors as well as to perform spatially resolved spectroscopy is reviewed by Lynn Gladden, Michael Mantle, and Andrew Sederman (University of Cambridge). This method allows even unsteady-state processes to be studied because of the rapid data acquisition pulse sequence methods that can now be used. In addition, MRI can be used to study systems with short nuclear spin relaxation times—e.g., to study coke distribution in catalytic reactors. 

A number of pulse-sequence methods are available for measurement of Ti values, and those most commonly used are the methods of saturation recovery (s.r.F.t.),69,70 progressive saturation (p.s.F.t.),71 inversion recovery (i.r.F.t.),72 and the Freeman-Hill modification of in-... 

D. Generic Problems with Iterative Multiple Sequence Methods. 154... 

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. 

Genotypic resistance assays use DNA sequencing methods to examine the reverse transcriptase and protease regions of fhe HIV genome for all resistance-associated mutations. A major drawback of fhis fesfing mefhod is that results are difficult to interpret and expert consultation is necessary. 

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

In the 1960s, scientists developed methods that allowed them to figure out the order, or sequence, of bases in a DNA molecule. The methods have been modified over the years so that machines can now do much of the work, but the principle remains the same. The sequencing method requires ... 

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