FIND ARTICLE

Volume: 
Issue: 
4
Date of issue: 

TILLING ( Targeting Induced Local called Lesions IN Genomes ) and FOX- hunting ( called Full- length cDNA Over- expressing gene hunting system ) is a new research methods that allow a massive and rapid functional analysis of genes. Acceleration work in this area of the biological sciences is necessary to structure the data obtained in genome sequencing programs , genes and ESTs ( ang.Expressed Sequence Tag) , as well as in research programs of gene expression changes , carried out using a DNA microarray analysis . Classical methods for functional studies have been and are carried out by " the characteristics of the gene " (called Top-down / forward ) . First, using the methods qualitative or quantitative mapping sought genetic markers characteristic of strongly coupled . Kosegregujące of markers characteristic is applied to a physical contig map of genomic libraries of clones in order to select a clone containing the gene sought , which is checked in the complementation assay . A positive result of this test confirms the intended biological function of the gene test , but the lack of expected phenotypic changes in the transformants is not the negation of the assumptions , due to the frequently occurring gene silencing . Another type of solutions for functional analysis of genes is the way " of mutations to the gene " (called Botom-up/Reverse ) , assuming the production , exploration and analysis of mutants and this is the approach used in both of these techniques . TILLING technique is a combination of traditional chemical mutagenesis with modern and sensitive technique to identify mutants point , SNP ( Single Nucleotide Polymorphism called ) , based on PCR analysis . This method involves the multiplication of a mixture of equal amounts of DNA of each of the mutants by PCR , and then the digestion products Customs1 endonuclease that recognizes the unpaired base in double-stranded DNA. An important and difficult to obtain a preliminary stage a sufficiently large and sufficiently saturated with the population of mutants , which depends both on the chemical agent and the genome to be modified . A second critical step is to prepare the template DNA that is collectible in 96 -well microplates. For the single PCR of DNA prepared mixture of 8 tubes for collecting DNA from individual rows of holes , then (up to 12 tubes ) DNA with subsequent columns of holes , so that material from one microplate can be analyzed in 20 ( 8 + 12 ) instead of 96 reactions. DNA analysis of many plates can further improve the collecting DNA from the same wells of the various plates . In order to identify each of the mutant PCR product is subjected to denaturation and annealing , resulting in the formation of heteroduplexes from the unpaired bases at the site of mutation. Digestion of the DNA endonuclease Customs1 specific to mismatched bases allows them to be identified after gel electrophoresis sequencing . The TILLING method to identify mutants is faster than classic method to identify mutants of the SNP , because i / instead koszto - and time-consuming sequencing using the restriction enzyme digesting renatured PCR products in mismatched nucleotides and standard DNA electrophoresis ; ii / PCR reaction is carried out on a large scale , on the template DNA which is the mixture of several / several samples . FOX- hunting technique , a new method of plant gene overexpression , which allows for quick isolation and sequencing in parallel with the functional analysis . It is a modification / development Arabidopsis transformation method for binary vectors introduced into plant cells by A. tumefaciens , by dipping in the slurry young inflorescences binary library . The binary vectors used in the FOX hunting system between the border sequences include : i / two tandem repeats of a transcription enhancer ( -490 to -90 sequence of the pre -bp promoter of the cauliflower mosaic virus 35S CaMV ) ; ii / 35S promoter ( -90 to -1 sequence bp CaMV ) ; iii / the sequence W - 5 ' leader , untranslated RNA segment of tobacco mosaic virus ( TMV ) , increasing the efficiency of translation of the introduced genes ; iv / cassette containing the gene , which we want to transform A. thaliana surrounded primer sequences ( GS4 and GS6 ) to facilitate the identification of the mutant and the sequence recognized by the endonuclease SfiI ; v / transcription terminator of the nopaline synthase gene (nos ) of the Ti plasmid and vi / selection gene for hygromycin resistance . To construct a binary vector library is used with a single copy of each gene collected in cDNA libraries constructed in the Lambda ZAP vectors, Lambda FLC - 1 -B. Advantages of the FOX- hunting are: and / small percentage of co-suppression of genes as a result of the use of full-length cDNA clones and standardized libraries in binary vectors ; ii / numerical limitation of the expression of housekeeping genes ( called house -keeping genes ) in normalized libraries ; iii / facilitate the analysis of the phenotype resulting from the short life cycle of A. thaliana ; iv / fairly easy isolation and sequencing of genes. The disadvantage of this system is the only limitation to the functional analysis of genes used to construct a library in A. tumefaciens . Both techniques , TILLING - FOX hunting and allow to accelerate genetic analysis . Programs that use it can also be an additional source of diverse material for breeding. The TILLING for this reason that it has been developed to study chemically induced mutants , allows immediate and direct introduction of obtained materials for breeding. The FOX- hunting perspective opens mainly in functional analysis , generating valuable mutants , in which overexpression of genes.

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The Editorial Board
Andrzej Łukaszyk - przewodniczący, Zofia Bielańska-Osuchowska, Szczepan Biliński, Mieczysław Chorąży, Aleksander Koj, Włodzimierz Korochoda, Leszek Kuźnicki, Aleksandra Stojałowska, Lech Wojtczak

Editorial address:
Katedra i Zakład Histologii i Embriologii Uniwersytetu Medycznego w Poznaniu, ul. Święcickiego 6, 60-781 Poznań, tel. +48 61 8546453, fax. +48 61 8546440, email: mnowicki@ump.edu.pl

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