遗传学实验技术

The major pathway for the removal of oxidative base damage is the DNA base excision repair pathway, found in prokaryotes and eukaryotes (1). In this pathway, oxidized DNA bases are removed by specific DNA glycosylases, leaving apurinic/apyrimidinic (AP) sites in the DNA (1,2). AP sites can also ari ...

DNA repair can occur by a variety of mechanistically distinct pathways . Recombinational DNA repair is one such pathway, and it requires the coordinated action of many different enzymes. In the best studied organism, Escherichia coli, more than 20 different proteins are involved . The recomb ...

The RecBCD enzyme serves two functions in the bacterial cell: it is a nuclease that destroys linear double-stranded DNA (dsDNA), and a DNA helicase that generates single-stranded DNA (ssDNA) used by RecA protein to initiate homologous recombination (1–3). A specific DNA sequence called Chi (5 ...

The analysis or manipulation of small segments of DNA in plasmids has long been routine, but standard methods fail or are very time consuming when applied to long segments or complex mixtures of DNA. At present, RecA protein-based techniques usually have been used for making defined changes in lar ...

The detection of single-base change mutations and polymorphisms is of enormous importance, both in research and in diagnostics. The ability to identify and score single nucleotide polymorphisms (SNPs) is becoming a key element of gene identification and mapping, and the future of human d ...

The use of resolvase enzymes to detect mutations (1) was developed in response to the demand for a method that could screen kilobase lengths of DNA for single nucleotide changes and small insertions and deletions. The method is a more simple, nontoxic alternative to the chemical cleavage of the mis ...

Three methods are outlined for finding protein genes and one for locating tRNA genes, as are routines for finding open reading frames and displaying the positions of stop codons. All the methods are contained in the program NIP. The correct interpretation of the analyses presented requires a go ...

The program PIP contains several ways of defining and searching for motifs (1,2). Searches for exact matches and percentage matches, the use of score matrices, and the creation and use of weight matrices are described here. All of the searches produce both listed and graphical output.

This chapter describes the use of routines for plotting hydrophobicity, charge, and hydrophobic moments, drawing helix wheels and predicting secondary structure. Use of all these routines is very straightforward, and they are contained in the program PIP.

Described here is one of the most powerful facilities provided by the program PIP: the ability to define and search sequences or libraries of sequences for complex patterns of motifs. Another chapter gives details of searching for individual motifs, but this chapter shows how to create indivi ...

The Staden-Plus software is an old menu-driven version of Rodger Staden’s software for manipulation of DNA and protein sequences that will run on an IBM compatible PC. This program is of use to those workers who do not have access to the newer versions of these programs, which are described in greater det ...

This chapter describes methods for comparing and aligning pairs of nucleic acid or protein sequences. The program described (SIP), the original version of which was first described in 1982 (1), is based around several methods for producing “dot matrix” plots and includes routines for assess ...

MacVector™, from Laboratory and Research Products, Eastman Chemical Company, New Haven CT, is an integrated comprehensive sequence analysis program. It provides the most commonly used nucleic acid and protein analyses, semiautomatic entry of sequence data from autoradiograms u ...

MacVector™ uses a variation (1) of the Wilbur-Lipman-Pearson algorithm (2–5) to find a “best” pairwise alignment between a single query sequence in memory and one or more other sequences stored in a folder on disk. The algorithm uses three comparison steps. A very rapid technique called hashing is u ...

When looking for similarities between two sequences, a matrix comparison is the method of first choice. A matrix analysis is unsurpassed for obtaining an overall picture of how the sequences are related, and it can detect significant features that other methods may miss.

One of the goals of molecular biology is to be able to deduce the three-dimensional structure of a protein directly from its amino acid sequence. As a first step toward solving this problem, a number of empirical methods have been developed for predicting a protein’s secondary structure and for appr ...

Locating restriction enzyme recognition sites is one of the most commonly used functions of sequence analysis software. MacVector™’s implementation is convenient and flexible, allowing the results of the search to be viewed interactively in many different ways. In addition to findi ...

Secondary structure prediction for RNA is fundamentally different from three-dimensional molecular modeling (1). A secondary structure of an RNA molecule is simply a collection of predicted base pairs subject to a few simple rules. Base pairs can be either G-C or A-U Watson-Crick pairs, or the w ...

Profile analysis uses information from a group of aligned sequences to create a generalized probe, the profile, for sequence or structural motifs. The profile contains information about the location and type of sequence conservation observed in the aligned sequences, the strength of the ...

Protein sequence similarities offer a convenient means for the classification and identification of protein families and superfamilies. Frequently, proteins descended from a common ancestor preserve their basic three-dimensional conformations even when they have accu ...