Sequencing templated method

This chapter describes various methods for generating templates suitable for sequencing from PCR products. Three general approaches are discussed (1) the cloning of PCR products, (2) preparation of single-stranded templates from PCR products, and (3) direct use of double-stranded PCR products as sequencing templates. 

The method we have used most for generating DNA sequencing templates is unbalanced priming, which utilizes asymmetric ratios of primer concentration to synthesize an excess of one strand during the PCR.11 This... 

Homologue(s) of known structure (template(s)) determined by database search methods (BLAST, PSI-BLAST, intermediate-sequence-search methods, HMM s, fold recognition methods)... 

Deciding how much of the template structure to remove before loop building depends on examination of the sequence alignment and the template structure. Sequence alignments with insertions and deletions are usually not unambiguous. Most sequence alignment methods ignorant of... 

A comparison of the alignment score of the target sequence with (many) different template proteins can be the basis of rating the significance of the produced structure model. P-values are a version of this approach that also applies to sequence analysis methods (see Chapter 2 of this volume), for which is has been studied extensively [9, 11, 70, 71]. In addition, the resulting models can be subjected to a wide variety of plausibility tests in order to ascertain their protein-likeness. Many of these checks will be made on the full-atom model of the protein that results from the procedures described in Chapter 5 of this volume. 

When RNA is to be sequenced, the method of choice is not to analyze the RNA itself but to use the methods of DNA sequencing on a DNA complementary (cDNA) to the RNA in question. The cDNA, in turn, is generated by using the enzyme reverse transcriptase, which catalyzes the synthesis of DNA from an RNA template. 

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