14-05-2012, 05:33 PM
Bioinformatics Resource Portal
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ExPASy is the new SIB Bioinformatics Resource Portal which provides access to scientific databases and software tools in different areas of life sciences including proteomics, genomics, phylogeny, systems biology, population genetics, transcriptomics etc. (see Categories in the left menu). On this portal you find resources from many different SIB groups as well as external institutions. The mission of UniProt is to provide the scientific community with a comprehensive, high quality and freely accessible resource of protein sequence and functional information. UniProt is comprised of four components, each optimised for different uses. The UniProt Knowledgebase (UniProtKB) is the central access point for extensive curated protein information, including function, classification, and cross-reference. It consists of two sections: UniProtKB/Swiss-Prot which is manually annotated and is reviewed and UniProtKB/TrEMBL which is automatically annotated and is not reviewed. The UniProt Reference Clusters (UniRef) databases provide clustered sets of sequences from the UniProtKB and selected UniProt Archive records to obtain complete coverage of sequence space at several resolutions while hiding redundant sequences. The UniProt Archive (UniParc) is a comprehensive repository, used to keep track of sequences and their identifiers. The UniProt Metagenomic and Environmental Sequences (UniMES) database is a repository specifically developed for metagenomic and environmental data.
Database Description for PIR-PSD
Release 80.00 (31 Dec 2004) is the final release for the PIR-International Protein Sequence Database (PIR-PSD), the world's first database of classified and functionally annotated protein sequences that grew out of the Atlas of Protein Sequence and Structure (1965-1978) edited by Margaret Dayhoff. Produced and distributed by the Protein Information Resource in collaboration with MIPS (Munich Information Center for Protein Sequences) and JIPID (Japan International Protein Information Database), PIR-PSD has been the most comprehensive and expertly-curated protein sequence database in the public domain for over 20 years. In 2002, PIR joined EBI (European Bioinformatics Institute) and SIB (Swiss Institute of Bioinformatics) to form the UniProt consortium. PIR-PSD sequences and annotations have been integrated into UniProt Knowledgebase. Bi-directional cross-references between UniProt (UniProt Knowledgebase and/or UniParc) and PIR-PSD are established to allow easy tracking of former PIR-PSD entries. PIR-PSD unique sequences, reference citations, and experimentally-verified data can now be found in the relevant UniProt records. Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule. In general, this form of mutagenesis requires that the wild type gene sequence be known. It is commonly used in protein engineeringHistory
Early attempts at mutagenesis were non-site specific using radiation or chemical mutagens.[1] Analogs of nucleotides and other chemicals were later used to generate localized point mutations,[2] examples of such chemicals are aminopurine,[3] nitrosoguanidine,[4] and bisulfite.[5] Site-directed mutagenesis was achieved in 1973 in the laboratory of Charles Weissmann using N4-hydroxycytidine which induces transition of GC to AT.[6][7] These methods of mutagenesis however are limited by the kind of mutation they can achieve.
In 1971, Clyde Hutchison and Marshall Edgell showed that it is possible to produce mutants with small fragments of phage ϕX174 and restriction nucleases.[8][9] Hutchison later produced with his collaborator Michael Smith in 1978 a more flexible approach to site-directed mutagenesis by using oligonucleotides in a primer extension method with DNA polymerase.[10] For his part in the development of this process, Michael Smith later shared the Nobel Prize in Chemistry in October 1993 with Kary B. Mullis, who invented polymerase chain reaction.
Basic mechanism
The basic procedure requires the synthesis of a short DNA primer which is complementary to the template DNA around the site where the mutation is to be introduced. The mutation may be a single base change (a point mutation), deletion or insertion, containing the desired mutation such as a base change. This synthetic primer is complementary to the template DNA around the base change so it can hybridize with the DNA containing the gene of interest. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene. The gene thus copied contains the mutated site, and is then introduced into a host cell as a vector and cloned. Finally, mutants are selected.
The original method using single-primer extension was inefficient due to a lower yield of mutants. The resulting mixture may contain both the original unmutated template as well as the mutant strand, producing a mix population of mutant and non-mutant progenies. The mutants may also be counter-selected due to presence of mismatch repair system which favors the methylated template DNA. Many approaches have since been developed to improve the efficiency of mutagenesis.
Approaches in site-directed mutagenesis
A large number of methods are available to effect site-directed mutagenesis,[11] although most of them are now rarely used in laboratories since the early 2000s as newer techniques allow for simpler and easier way of introducing site-specific mutation into genes.
[edit] Kunkel's method
In 1987 Kunkel et al. introduced a technique which reduces the need to select for the mutants.[12] The vector DNA to be mutated is inserted into an E. coli strain deficient in two enzymes, dUTPase and uracil deglycosidase. The dUTPase deficiency prevents the breakdown of dUTP, a nucleotide that normally replaces dTTP in RNA, resulting in an abundance of dUTP; the uracil deglycosidase deficiency prevents the removal of uracil from newly-synthesized DNA. As the double-mutant E. coli replicates the vector DNA, its enzymatic machinery may therefore misincorporates dUTP instead of dTTP, resulting in DNA which contains some uracils. This copy is extracted, and then used as template for mutagenesis. An oligonucleotide containing the desired mutation is use for primer extension. The heteroduplex DNA formed may be chimeric containing one strand unmutated and containing UTP, and the other strand mutated and containing dTTP. The DNA is then first treated with uracil deglycosidase which removes the uracil in the template, then with alkali which degrades the template DNA as it has its uracil removed making it sensitive to alkali. The resulting DNA therefore consists of only the mutated strand.
Cassette mutagenesis
Unlike other methods, cassette mutagenesis need not involve primer extension using DNA polymerase. In this method, a fragment of DNA is synthesized, and then inserted into a plasmid.[13] It involves the cleavage by a restriction enzyme at a site in the plasmid and subsequent ligation of a pair of complementary oligonucleotides containing the mutation in the gene of interest to the plasmid. Usually the restriction enzymes that cuts at the plasmid and the oligonucleotide are the same, permitting sticky ends of the plasmid and insert to ligate to one another. This method can generate mutants at close to 100% efficiency, but is limited by the availability of suitable restriction sites flanking the site that is to be mutated.