REtools: A laboratory program for restriction enzyme work: enzyme
Contents
1. BMC Bioinformatics 2006 7 98 Administration Figure 2 http www biomedcentral com 1471 2105 7 98 Home page where the different functions of REtools can be selected 3 Three files called BRLBuffers MBIBuffers and NEBBuffers list the recommended buffer for the differ ent restriction enzymes carried by these manufacturers 4 The LabList file is a personalized list which regroups the restriction enzymes present in one s own laboratory Results REtools a FileMaker Pro based Runtime solution is a rela tional database built around 7 files The organization of these files are depicted in Fig 1 The main organizer file called EnzymesLabo located in the center directly han dles administration of the lab list as well as user requests Directly linked to this central file are 6 auxiliary files con taining different data necessary to process users requests 1 The AllREnar file which contains all known enzymes as provided by REbase 2 as well their specificities such as recognition sequence cleavage optimal digestion tem perature heat inactivation data methylation and mini mal number of flanking nucleotides to allow digestion 2 The Protos file contains an exhaustive list of all isoschizomers and neoisoschizomers taken from the All REnar file 3 Three files called BRLBuffers MBIBuffers and NEBBuffers list the recommended buffer for the differ ent restriction enzymes carried b2. 05 7 98 e sensitivity to methylation with each possible methylase action and their corresponding effect on cleavage being specified e potential isoschizomers identical site and cleavage present within the laboratory enzymes e number of flanking nucleotides required for cleavage for example useful when designing PCR primers harbor ing restriction sites at their extremities Perform multiple digestions This section is of great interest to determine optimum reaction conditions for the chosen enzymes when multi ple digestions are performed The user selects the enzymes to be used for the multiple digestions from the pop up lab list REtools analyzes the enzymes selected and addition ally proposes isoschizomers when available by default This is important since there may be cases where two enzymes do not share common reaction conditions whereas the isoschizomers would REtools also indicates that neoisoschizomers identical site but distinct cleav age are available but does not include them in the anal ysis except if the user chooses to since the cleavage pattern is different from the originally selected enzyme A warning is also displayed in case some of the selected enzymes may be affected by methylation Once the choice of enzyme has been validated a screen on which all infor mation related to the multiple digestions is displayed Fig 3 Find information on an enzyme not present in the lab This is
3. 2 BioMed Central Software REtools A laboratory program for restriction enzyme work enzyme selection and reaction condition assistance Patrick Martin Kim E Boulukos and Philippe Pognonec BMC Bioinformatics Address CNRS UMR 6548 Parc Valrose Universit de Nice Sophia Antipolis Nice France Email Patrick Martin pmartin unice fr Kim E Boulukos boulukos unice fr Philippe Pognonec pognonec unice fr Corresponding author Received 03 October 2005 Accepted 28 February 2006 Published 28 February 2006 BMC Bioinformatics2006 7 98 doi 10 1 186 1471 2105 7 98 This article is available from http www biomedcentral com 47 I 2105 7 98 2006Martin et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License http creativecommons org licenses by 2 0 which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited Abstract Background Restriction enzymes are one of the everyday tools used in molecular biology The continuously expanding panel of known restriction enzymes several thousands renders their optimal use virtually impossible without computerized assistance Several manufacturers propose on line sites that assist scientists in their restriction enzyme work however none of these sites meet all the actual needs of laboratory workers and they do not take into account th
4. 8 Inhibition BRL MBI NEB sheath Required NEE Hew England Biolibs Additional information 4 Additional information Possible OF 3 Additional information 1 4 Additional information 2 Additional information o H Additional information Possible 4 Bt 4 Additional information Possible 4 Additional information 3 Additional information 2 4 Additional infomation Possihle 4 Additional information o 2 Additional information 3 w U Additional information Always 2 G j Additional information fF O f Additional information 3 oo 3 Additional information 2 wH Additional information 3 Additional information 2 Additional infornation _ oF Additional information 2 3 Additional information Possible B Additional information http www biomedcentral com 1471 2105 7 98 Home Enzyme Site Prototype Optimal 37 C F F F activity activity Aas GACHNNHH NNGTC Drdl mC AatIT GACGT C Aatl a7 C Acc6s G OTAC Aspllal 47 C Accl GT MEAC Accl 27 C ATII CI TTAAG Af are Alw44I G TGCAC pall 47 C Apal GGGCC C Anal fa 100 Ascl GG CGCECe Ascl T C Asel AT TAAT Vepl cha Aval C YOGRG Awal 37 C Avall G GW Avvall a7 C Avril C CTAGG Avr 47 C BamHI G GATCC BamHI HC Bell T GATCA Bell so c 50 Ball GCCANNN HGGG Bell a7 C BallIl 4 GATCT Bell a Epul1021 GC THAG Fspl SPC BsaAl YAC GTR Bead a7 C BseR I GAGGAGIIOS BseRI a7 C EshTI 4 CCGGT Agel 47 C Bsml GAATGOILL Barr ae 40 Bsp1201 G GGCCl Bspl2ol 37 C Figur
5. ction enzymes and all searches in REtools indicate whether the chosen enzymes or equivalents are availa ble in the user s own laboratory The interface has been designed to make its use as straightforward as possible and no training is required to use REtools On line help is available throughout the program in case additional information is needed REtools was developed using File Maker Pro It is freely available to the academic commu nity upon request and does not request a FileMaker Pro license since it is distributed as a bound solution Implementation REtools a FileMaker Pro based Runtime solution is a rela tional database built around 7 files The organization of these files are depicted in Fig 1 The main organizer file called EnzymesLabo located in the center directly han dles administration of the lab list as well as user requests Directly linked to this central file are 6 auxiliary files con taining different data necessary to process users requests 1 The AllREnar file which contains all known enzymes as provided by REbase 2 as well their specificities such as recognition sequence cleavage optimal digestion tem perature heat inactivation data methylation and mini mal number of flanking nucleotides to allow digestion 2 The Protos file contains an exhaustive list of all isoschizomers and neoisoschizomers taken from the All REnar file Page 2 of 8 page number not for citation purposes
6. e 4 List of the restriction enzymes present in the laboratory include any newly described enzymes reported in REbase and or restriction enzyme company catalogs The com mercial availability of each enzyme from 19 different companies is indicated REtools users can update their AllREnar files by simply redownloading and rerunning the REtools installer Find an enzyme in our lab list This function is used to access all the information availa ble for an enzyme present in one s personalized labora tory enzyme list In addition to the name and recognition site optimal digestion temperature and optimal buffers from 3 companies Invitrogen BRL Fermentas MBI and New England Biolabs NEB are indicated as well as the percentage of activities in the other buffers More specific characteristics of the selected enzyme are proposed when available e heat inactivation conditions when applicable This is of interest when the researcher wants to kill enzyme activity before performing subsequent reactions in the same tube without having to worry about the first enzyme s activity e time temperature and buffer conditions as recom mended by the manufacturers Page 5 of 8 page number not for citation purposes BMC Bioinformatics 2006 7 98 BEL besttrogzer MEI Fennertas HEE Hew England Biolabs Go Figure 5 Search of restriction enzymes sharing similar reaction conditions http www biomedcentral com 1471 21
7. e enzymes actually present in one s own laboratory Results Using FileMaker Pro we developed a stand alone application which can run on both PCs and Macintoshes We called it REtools for Restriction Enzyme tools This program which references all currently known enzymes gt 3500 permits the creation and update of a personalized list of restriction enzymes actually available in one s own laboratory Upon opening the program scientists will be presented with a user friendly interface that will direct them to different menus each one corresponding to different situations that restriction enzyme users commonly encounter We particularly emphasized the ease of use to make REtools a solution that laboratory members would actually want to use Conclusion REtools a user friendly and easily customized program to organize any laboratory enzyme stock brings a software solution that will make restriction enzyme use and reaction condition determination straightforward and efficient The usually unexplored potential of isoschizomers also becomes accessible to all since REtools proposes all possible enzymes similar to the one s chosen by the user Finally many of the commonly overlooked subtleties of restriction enzyme work such as methylation requirement unusual reaction conditions or the number of flanking bases required for cleavage are automatically provided by REtools Background Restriction enzymes are incontestably the most funda mental t
8. e the restriction enzymes availa ble in one s laboratory Fig 4 This list can be sorted according to enzyme name nucleotide site or prototype name Recommended digestion temperatures are indi cated as well as the percentage of activity at 37 C if the recommended temperature is different If applicable inhibition by or requirement for methylated DNA is men tioned Finally recommended buffers are noted for the three major restriction enzyme companies Further infor mation on any enzyme is accessible directly from this list Find enzymes sharing reaction conditions This function of REtools aims at finding among all known enzymes those which share selected reaction conditions and or specificities such as methylation sensitivity This is of particular interest when an already digested sample must be redigested to separate comigrating fragments By entering the reaction conditions the list of compatible enzymes will be displayed allowing the scientist to select the one s that will correspond best to the situation Discussion Organization is an important part in efficiently running a laboratory Quite often the high turnover of students and postdoctoral fellows associated with dynamic laboratories makes tracing and archiving biological samples and rea gents quite difficult In order to reduce these problems we set up to develop laboratory oriented applications based on FileMaker Pro The originality of these applications is tha
9. erating system s Windows and MAC Programming language FilemakerPro Other requirements None License GNU GPL Any restrictions to use by non academics None Authors contributions The structure of the program was mainly developed by P Martin and the interface was mostly the work of P Pog nonec with the help of K E Boulukos Acknowledgements This work was made possible by the support of CNRS and Proskelia Pros trakan References I NEBCutter http tools neb com NEBcutter2 index php 2 Official REBASE Homepage http rebase neb com rebase rebase html 3 Restriction Enzymes Ressource __ http www promega com guides re_guide EnzymeX http www mekentosj com enzymex Zaldumbide A Boulukos KE Pognonec P Virtual nitrogen tank to monitor frozen cell stocks Biotechniques 2000 29 122 126 UA http www biomedcentral com 1471 2105 7 98 Boulukos KE Pognonec P MICE a program to track and moni tor animals in animal facilities BMC Genet 2001 2 4 Roche RE Finder http www roche applied science com fst products htm benchmate Publish with BioMed Central and every scientist can read your work free of charge BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime Sir Paul Nurse Cancer Research UK Your research papers will be e available free of charge to the entire biomedical community e peer reviewed and published im
10. he Netherlands Cancer Institute responded to most of our needs This program does not however assist in multiple digestions involving more than two enzymes nor does it propose alternate iso neo schizomers when the selected enzymes are not present in one s customized laboratory list Finally EnzymeX exclusively runs on MacOSX 10 2 or later versions which excludes its use in laboratories run ning on Windows For these reasons we designed REtools whose philoso phy relies first on the optimization of the laboratory enzyme stock usage and second on a user friendly and intuitive program interface so that people really work with it REtools provides information on both individual enzyme reaction conditions and on multiple digest con ditions REtools also allows users to find alternative enzymes that may be present in the laboratory if the enzyme originally chosen is missing as well as determin ing all possible enzymes able to recognize a selected restriction site REtools will make the chore of browsing restriction enzyme catalogs in the hope of finding the right piece of information a thing of the past REtools can be distributed to all laboratory members since it is provided as a stand alone application that does not require any third party license to run To facilitate handling of the enzyme lab stock an administrator can be designated who will be in charge of customizing REtools to the enzymes present in the laboratory The file c
11. mediately upon acceptance e cited in PubMed and archived on PubMed Central e yours you keep the copyright Submit your manuscript here O BioMedcentral http www biomedcentral com info publishing_adv asp Page 8 of 8 page number not for citation purposes
12. o simultaneously digest DNA with several different restriction enzymes 4 Find information on an enzyme not present in the lab searches for information on a specific enzyme not present in the user s laboratory in particular to see if an equivalent enzyme isoschizomer could be available in the lab 5 Find enzymes corresponding to a sequence searches for enzymes recognizing a selected restriction site sequence 6 Display our enzyme lab list displays the personal ized list of enzymes present in one s lab which can be organized in the Administration section 7 Find enzymes sharing reaction conditions searches for enzymes whose activities are compatible with the selected reaction conditions buffer pH temperature Each of these 7 functions is presented below Administration This password protected access has been designed for lab oratories where restriction enzymes are under one per son s responsibility called the RE administrator The RE administrator can choose from the more than 3500 enzymes present in REtools to customize a REtools lab list to match enzymes actually present in one s laboratory This customized lab list can be printed out and subse quently posted on a freezer door More than 3500 enzymes are recorded in the AllREnar file present in the REtools folder We will update this list twice a year to Page 4 of 8 page number not for citation purposes BMC Bioinformatics 2006 7 9
13. ool used in molecular biology Over the past 40 years more than 3500 restriction enzymes have been iso lated from hundreds of strains of bacteria It is the sequence specificity of enzymes which renders them so valuable However because of the large number of restric tion enzymes now available on the market it is becoming quite difficult to navigate through an ever increasing choice to find the most suitable enzyme that one actually needs One of the drawbacks is the excessive simplifica tion now seen in many laboratories when it comes to digesting DNA Quite often researchers tend to focus on the most commonly used restriction enzymes found in Page 1 of 8 page number not for citation purposes BMC Bioinformatics 2006 7 98 the majority of polylinkers and are afraid to use the so called exotic enzymes which may not work as well Frequently researchers are not aware of the potential of isoschizomers recognizing and digesting the same DNA sequence once again mainly because of the huge panel of enzymes currently available Indeed approximately 330 different DNA motifs are recognized by restriction enzymes each of them being cut by a prototype enzyme But in addition to that prototype an average of ten other restriction enzymes recognize the same DNA sequence This is potentially of great interest when a site is to be used to insert an incompatible fragment For example Apal is a widely known and frequently used restric
14. ratory enzyme list by the administrator http www biomedcentral com 1471 2105 7 98 time browsing through different manufacturers catalogs or web sites Some software have been developed which greatly help molecular biologists including NEB s NEB Cutter 1 and their well known and exhaustive REbase 2 and Promega s Restriction Enzymes Resource 3 However these programs have their limitations In partic ular they do not take into account the enzymes actually available in the user s own laboratory In addition NEB Cutter requires the user to enter the nucleotide sequence to be digested and thus does not provide information on restriction enzymes per se REbase is exclusively an ency clopedia like description of all known restriction enzymes while Promega s application is oriented toward restriction enzymes and buffers specifically distributed by this manufacturer Recently an attractive program called EnzymeX has been developed 4 Unfortunately this program only runs on MacOSX10 2 or later versions and does not provide any information regarding isoschizomer use In order to bypass these limitations we developed a stand alone application that permits the researcher to find all the information available on all known restriction enzymes gt 3500 as well as their cutting characteristics and inter compatibilities in just a few clicks Importantly REtools is also a way to monitor one s laboratory panel of restri
15. reated lab list during this process can then be distributed to all users who will then work with the actual enzymes present in laboratory Page 7 of 8 page number not for citation purposes BMC Bioinformatics 2006 7 98 REtools and its user manual pdf see Additional file 1 are available to the academic community free of charge upon request to pognonec unice fr Please enter REtools in the subject field Conclusion REtools is a user friendly program for organizing labora tory enzyme stocks We designed it to make restriction enzyme use and reaction condition determination straightforward and efficient In particular we made the usually unexplored potential of isoschizomers accessible to all Finally many of the commonly overlooked subtle ties of restriction enzyme work are automatically provided by REtools REtools is a stand alone application and thus can be distributed to all laboratory members without any requirement for a third party license To facilitate han dling of the enzyme lab stock an administrator can be designated who would be in charge of customizing REtools with the enzymes present in one s own labora tory REtools and its user manual pdf are available to the academic community free of charge upon request to pognonec unice fr Please enter REtools in the subject field Availability and requirements Project name REtools Project home page http www unice fr LPCM soft ware html Op
16. similar to Find an enzyme in our lab list except that all known enzymes are accessible Importantly REtools will directly link the searched enzyme to isoschi zomers same site and same cleavage and neoisoschi zomers same site but distinct cleavage that may be present in one s personalized laboratory list This is quite Page 6 of 8 page number not for citation purposes BMC Bioinformatics 2006 7 98 useful since other enzymes present in one s laboratory may be perfectly compatible with the planned digestion but unknown to the scientist Find enzymes corresponding to a sequence This section of REtools is designed to determine all the existing restriction enzymes recognizing a specific nucle otide sequence The prototype enzyme first discovered restriction enzyme for a specific site and cleavage for the chosen sequence is listed as well as all the isoschizomers commercially available All other prototypes matching the same nucleotide sequence but with a distinct cleavage pattern are also displayed Further inquiries on these pro totypes can be made by clicking on their names to find out for example if they or an isoschizomer are present in one s personalized laboratory list Additionally REtools indicates the effects that common modifying enzymes like Klenow or Mung Bean nuclease would have on the DNA extremities following digestion with the chosen enzyme Display our enzyme list This is to directly visualiz
17. t they were created to organize daily events in the lab oratory rather than forcing users to adapt to the software The two first applications so far developed and published by our laboratory FCSM for the monitoring and organi http www biomedcentral com 1471 2105 7 98 zation of samples frozen in liquid nitrogen or in freezers 5 and MICE for the management of mice projects 6 obtained a very positive feedback from the scientific com munity In the recent years we became more and more aware of the problems and limitations in restriction enzyme usage Sci entists cannot reasonably keep up with the permanently growing panel of potential alternative solutions when it comes to selecting a restriction enzyme for a particular digestion We thus looked for a program to assist scientists both in the selection process among enzymes present in the laboratory as well as in the choice of optimal reaction conditions for the restrictions planned To our astonish ment very few solutions were available and most of these were restricted to reagents sold by their manufacturers such as the previously described NEB s NEBCutter 1 and Promega s Restriction Enzymes Resource 3 Another program RE Finder 7 from Roche is limited to 339 enzymes and does not help with multiple digestions None of these three software allow following a personal ized laboratory enzyme list Only one program EnzymeX 4 developed by molecular biologists in t
18. tion enzyme which cuts the sequence GGGCC C But an Apal digested fragment is only compatible with another Apal digested fragment However if one uses Bsp120I or BspOMI both commercially available and cut G GGCCC instead of Apal then the obtained extremities can be religated with NotI digested fragments GC GGCCGC without any further molecular tinkering This is not an isolated situation since other well known enzyme restriction sites can be cleaved with different isoschizomers such as KpnI with Asp718I or Smal with Cfr9I when a non blunt cut is needed Therefore it is quite difficult to be aware of all the possi bilities offered by the multiple restriction enzymes availa ble on the market and frequently one tends to restrict his her choice to the most commonly known restriction enzymes To take advantage of the vast panel of restriction enzymes commercially available one must spend a lot of AllREnar Protos lsoschizomers Isoneoschizomers All known restriction enzymes MBIBuffers MBI buffer information NEBBuffers LabList EnzymesLabo List of restriction enzymes present in the laboratory NEB buffer information Administration User interface BRLBuffers BRL buffer information Figure Structure of the REtools relational database Boxes represent the files constituting REtools Black arrows indicate data fluxes in response to user requests The red arrow repre sents customization of the labo
19. y these manufacturers 4 The LabList file is a personalized list which regroups the restriction enzymes present in one s own laboratory REtools proposes 7 main functions directly available from the opening screen Fig 2 1 Administration password protected to customize update the enzyme list to organize enzymes actually avail able in one s laboratory 2 Find an enzyme in our lab list searches for informa tion on a specific enzyme present in one s laboratory Page 3 of 8 page number not for citation purposes BMC Bioinformatics 2006 7 98 http www biomedcentral com 1471 2105 7 98 Additional information on each selected enzyme is displayed below BEL Eevitrogzer MEI Fenmertas HEE Her England Biolabs Methylation problem No Muthey etic AAGCTT amp AGCTT i basesrequiredtor 3 o Actrity ia 37 00w 2 S0Actiriny O 0 20 Figure 3 Optimum Butter Cleavage O BRL MBI NEB Yes Yes 65 C 65 C 20mm 20mn Crret lap Optimum Butter E Y 100 50 100 50 100 ll 40 Multiple digest screen Enzymes selected by the user are indicated as well as those chosen among them by REtools as the most suited for common conditions Optimum buffer s are indicated down to a minimum of 50 activity In addition information on each enzyme is visible in the lower panel 3 Perform multiple digestion searches for optimum conditions buffer pH temperature t
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