Document update: October 1998

1. What is CloneIt ?
2. What's new in this version ?
3. Download CloneIt
4. The main screen.
5. Quick start.
6. Bibliography
7. Acknwoledgment.

1. What is CloneIt ?

   
   Molecular biologists often have to sub-clone plasmidic vectors: a DNA plasmid is cleaved and ligated with an exogen DNA fragment previously excised from an other plasmid. The necessary cuts are achieved by restriction enzymes which then must be carefully choosen in order to minimize the steps required to obtain the desired molecule. During the selection of those enzymes, the main difficulties encountered come from: the knowldege of:
   • the enzymes' characteristics
   • the localization of the cuts within the sequence
   • the complementarity between the protuding ends
   • the possible self ligation of the vector
   • the use of modifying DNA polymerases that generate blunt ends
   • the constraint to clone the insert in-frame with a vector sequence
   • the use of partial digestions
   • the creation of a stop codon after the ligation.
   This exercise takes a long time even with a computorized help of the classic DNA analysis softwares, such as "DNA Strider" , that only help the user by localizing restriction sites that are present a few times in a plasmid sequence. All combinations cannot be humanly checked by the scientist, so a simple cloning strategy can be missed by the experimenter.
   A small history: In our laboratory, the problem was set with the requirement of making in-frame deletions after using the yeast two-hybrid system (Fields and Song, 1989). We had previously shown that the rotavirus non-structural protein NSP2 interacts with NSP5 and we wanted to generate various - and easy to make - in-frame deletions of those proteins. These deletions would allow us to map their regions of interaction and to discard the transactivating domain of NSP5 that creates false positive in the yeast two-hybrid system. A possible approach was the use of mutagenic PCR that creates restriction sites in a sequence which will be subcloned before being inserted in the desired plasmid. Another approach was the digestion of a plasmid vector by two enzymes excising a fragment in the coding sequence, followed by a ligation closing the digested vector and keeping the translation frame. We developed a program that quickly finds in-frame deletions using restriction enzymes and frameshifts (using digestion, fill-in and ligation) in a plasmid sequence, Then, as the main functions and procedures were being developed, we have extended the capacities of the program to find strategies to sub-clone a fragment from a plasmid to another vector while still controling the problems described above. This program is not an expert system, as it does not "learn" the logical steps accomplished by the biologist and it does not have to be accompanied in its search: it just runs an algorithm that explores all the possible enzymes combinations that could be used to clone the molecules.
   This program called CloneIt , written in ANSI C, provides a useful aid for any molecular biologist who wants to quickly find sub-cloning, in-frame deletions, frameshifts strategies, which would otherwise be difficult to discover.
   This program handle parameters such as
   • usage of a phosphatase (CIP)
   • usage of modifying polymerases that blunt overhanged ends
   • partial digestions
   • 5' or/and 3' in-frame ligation
   • digestion post ligation
   • digestion to direct insert
   • detection of stop codon after ligation
   • Compare 2 sequences from the point of view of restriction sites.
   • Display a restriction map.
   • Translation of restriction enzymes databases.
   • Management of large cloning projects.
   This is not the reviewed version of the program but a more sophisticated. The first version is available on request.

2. What's new in this version ?

   
   
   • CloneIt reads binary files from DNA Strider.
   • CloneIt finds silent mutations.
   • CloneIt handle the Enzyme's temperatures and buffers.
   • CloneIt provides a pathway to the GAP and BestFit GCG tools
   • CloneIt can now browse the strategies (next, previous..).
   • CloneIt reads the environment preferences to find a common REBASE file.

3. Download CloneIt

   
   CloneIt package contents (ASCII plain text):
   • cloneit.html
     this web page.
   • CloneIt.c
     the ANSI-C source code [what's that ?!].
   • allenz REBASE file (allenz *).
   • a VECTOR sequence.
   • an INSERT sequence.
   For old users: the "Polylinkers.Set" and the "Project" files are now automaticaly created by the new software version.

   Trade Mark: National Number 97/704582
   Institut National de la Propriete Industrielle
   26, rue de St Petersbourg 75800 Paris Cedex 08 FRANCE
   Order Number:18.NOV1997
   Copyright Notice: Permission to use, copy, modify, and distribute this software and its documentation is hereby granted, subject to the following restrictions and understandings:

   • 1) Any copy of this software or any copy of software derived from it must include this copyright notice in full.
   • 2) All materials or software developed as a consequence of the use of this software or software derived requires the express, written author permission.
   • 3) The software may be used by anyone for any purpose, except that its redistribution for profit or inclusion in other software sold for profit requires the express, written permission of the author.
   • 4) This software is provided AS IS with no warranties of any kind. In no event will the author be liable for any lost revenue or profits or other special, indirect and consequential damages.

   The CloneIt source code

   
   This is what is called the "source code of the program". This source is written in ANSI-C an international programming language. You will have to convert this text to a an application with a "compiler". The compilers, such as GNU gcc are often present on the UNIX machines. This program has been successfully tested on the GNU compiler gcc (version 2.6.2) but it should be compatible on any ANSI-C compiler. Nevertheless, read carefully the first lines of the source code, you may have to delete a line if you do not have PICO or LYNX on your station...
   To compile it, copy the file "CloneIt.c" on your UNIX directory. Then:
   • if your've got the PICO, LYNX... softwares on your UNIX station, type:

     gcc -oCloneIt CloneIt.c

   • if you haven't got those softwares, type:

     gcc -DNOT_UNIX -oCloneIt CloneIt.c

     
     
   • if you compile this program with an other compiler than gcc (ex: cc), add -D__GNUC__ to the command line
   
   this creates a program called "CloneIt".
   Type

   CloneIt

   
   to run the program

   

4. The main screen.

   
   

   ___________________________________________________ CloneIt V2.0 Pierrot LINDENBAUM Biologie Moleculaire des Rotavirus. VIM INRA. 78350 JOUY-EN-JOSAS. FRANCE. E.Mail:lindenb@biotec.jouy.inra.fr. ___________________________________________________ A command-line is available, type "CloneIt -HELP " for more information PROJECT. Run a project. VECTOR: Open a DNA sequence for VECTOR. Define VECTOR cloning box boundaries [875-906]. Define ATG position [875]. Set to antiparallele. View sequence.(UNIX only) pGBT9 restriction map. INSERT: Open a DNA sequence for INSERT. Define INSERT cloning boxes boundaries [880-1155] [3359-3634]. Define ATG position [882]. Set to antiparallele. View sequence.(UNIX only) pbs_RF2 restriction map. DELETIONS AND FRAMESHIFTS Find in frame deletions in INSERT. Look for Carboxy terminal deletions [X]. Find frameshifts in INSERT. Minimum percentage of insert [20 %] Maximum percentage of insert [80 %] SILENT MUTAION Finding silent mutation REBASE FILE: Open a REBASE File. [RebaseREL] (187 enzymes). Get information about an enzyme... Update (UNIX only) Enzymes Database Convertion... Show Rebase.(UNIX only) GCG Wisconsin Package © alignments (UNIX only): Gap (DNA). Gap (translated sequence) BestFit (DNA). BestFit (translated sequence) PARAMETERS: Display messages [X]. Speed optimization (Discard short sites) [X]. Use Klenow or T4 DNA Pol.[ ]. Number of partial digestion allowed = 1. Allow partial digestion only if enzyme blunt [ ]. Continue to search a solution within a couple [ ]. Allow non-overlapping overhangs [ ]. Use C.I.P. [ ]. Clone in frame at 5' of insert (NH2) [ ]. Clone in frame at 3' of insert (COOH)[ ]. Allow to use incompatible buffers[ ]. Allow to use incompatible temperatures[[ ]. CloneIt V1.2: Intersections. Find strategies to clone insert into vector. New Sequence. (UNIX only) Quit. Your choice /* logic choice */: If you use this program, please, cite it in your papers

   • Open a DNA sequence.

     
     The program takes as input plasmid sequence a DNA Strider or a FASTA sequence text file.
     The "VECTOR" plasmid, is designed to be the plasmid where the user wants to subclone a DNA fragment. This DNA fragment is excised from the "INSERT" plasmid.Since version 2, CloneIt can read binary files from DNA Strider. The sequence in FASTA format or DNA Strider (text ASCII) must be localized in the same folder than CloneIt.

     > DNA sequence pbs rf2 5895 b.p. complete sequence TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGAT GCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGA GCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAAT TGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCG GCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTA AAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATC AAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAA AGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACG (...) CTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAA GTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGC GAATTCGAGCTCGGTACCCGGGGCTATTAAAGGTTCAATGGCGTACAGGAAACGTGGAGCGCGCCGTGAGGCGAATATAA ATAATAATGACCGAATGCAAGAGAAAGATGACGAGAAACAAGATCAAAACAATAGAATGCAGTTGTCTGATAAAGTACTT TCAAAGAAAGAGGAAGTCGTAACCGACAGTCAAGAAGAAATTAAAATTGCTGATGAAGTGAAGAAATCGACGAAAGAAGA ATCTAAACAATTGCTTGAAGTTTTGAAAACAAAAGAAGAGCACCAAAAAGAGATACAATATGAAATTTTGCAAAAAACGA TACCAACATTTGAACCAAAAGAGTCAATATTGAAAAAATTGGAGGATATCAAACCGGAACAAGCGAAGAAGCAGACTAAG CTATTTAGAATATTTGAACCGAGACAGCTACCAATTTATAGAGCGAATGGTGAAAAAGAGTTGCGTAACAGATGGTATTG GAAGCTGAAGAAAGATACTTTACCAGATGGAGATTATGATGTTAGAGAATACTTTCTAAATTTGTATGATCAGGTTCTTA CTGAAATGCCAGATTATTTACTATTAAAAGATATGGCAGTTGAAAATAAAAATTCGAGAGATGCCGGTAAAGTTGTTGAT TCTGAAACAGCAAGTATCTGTGATGCTATATTTCAAGATGAGGAAACAGAAGGTGCAGTGAGAC

     Degenerate template are not allowed. Numbers will be discarded. ").The DNA sequence is a circular plasmid sequence. A short database of different sequence of classic oligonucleotides is used by the program to try to localize the insert bounds. Translated sequences are supposed to be oriented from NH2 to COOH. Input is case sensitive.
     

     -------------------------------------------------------------------------------- Input INSERT sequence name : Opening INSERT. 4737 bp in 'p5_1'. Your sequence seems to be a "T7/T3" type plasmid. The cloning boxes may be localized between 880 and 2476. The cloning box(es) boundaries and the ATG position have been searched, please check the displayed datas. --------------------------------------------------------------------------------

   • The Polylinkers file.

     
     This database is written as a text in the polylinkers file. At the first use of CloneIt a default file is created. If CloneIt can't recognize the plasmid, the user must input the positions of those extremities himself so he will have to determine them before running the program.

     ; CloneIt ; List of Polylinkers. ; ;do write the oligo that you would have used to sequence your plasmid ; syntax is "name, oligo1,oligo2," ; ; Baculo,ttttactgttttcgtaacagtttt,cggatttccttgaagagagta, pBK,ggtctatataagcagagctggt,acaggaaacagctatgaccttg, T7/T3,aattaaccctcactaaaggg,taatacgactcactataggg, pIIIMS2-1,ttccggctagaactagtggatcc,tcgactctagaggatcg, pIIIMS2-2,agagtcgacctgcaggcatgcaagctg,gctagaactagtggatcc, pGBT9,CAGTTGACTGTATCG,GCCCGGAATTAGCTTGG, pGAD424,CCAAAAAAAGAGAT,TTCAGTATCTACGATTCAT, pGADGL,CCAAAAAAAGAGATC,ACTATAGGGCGAATTGG,

   • Define cloning boxes boundaries.

     
     The VECTOR contains one cloning box ( a polylinker region ) where restriction sites should be localized to linearise it. Meanwhile the INSERT contains two cloning boxes where the endonucleases should free the DNA fragment. short database of different sequence of classic oligonucleotides is used by the program to try to localize the insert bounds, therefore it can easily localize the position of an insert in a pBS-like plasmid. If CloneIt cannot recognize the plasmid, the user will have to input the positions of those extremities himself.

   • Define ATG position.

     
     If the coding frame is of importance, the program can find the best frame to be used on each plasmid. This step may be usefull either to manage in-frame ligations or to test if a ligation creates a stop codon. In the INSERT plasmid, the best candidate is the largest Open Reading Frame (ORF) found inside the fragment. On the contrary, in the VECTOR plasmid, the best candidate is the largest ORF.

   • Restriction map.

     
     The program may also display a linear restriction map of any sequence which represent the two DNA strands, the translated six frames and the restriction sites on the very same screen.The first translated sequence is within the user's defined frame.

     SACI SALI ECORI : hincii styi acsi : acci ECO52I NCOI alwi : ECL136II eaei bstdsi xhoii : bsp1286i bsh1285i MSCI xmni BAMHI : banii : NOTI eaei ecorv : vspi alwi : alw21i : HINDIII: : : : : : :: : : : : :: TGGCCATGGATATCGGAATTAATTCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCA \ 5266 ¥ ¥ ¥ ¥ ¥ ¥ ¥ \ ACCGGTACCTATAGCCTTAATTAAGCCTAGGCTTAAGCTCGAGGCAGCTGTTCGAACGCCGGCGT \ 5330 T P T :I S E :L I R::I R: I R: A P :S T :S L :R P H -> :A :M D: I G :I: N S D: P N S S S V: D K: L A::A A L -> : H: G Y R N: * F G :S E :F E :L R R Q A C G: R T -> : P: V $ L R: L * A :$ A :* A :R P L Q E F A: P T <- :G :T G: I A :K: I L G: L S L S S A: A T: R V::G A H <- $ R Y :R Y G :* N L::R P: K L: E L :C S :N S :R R R <- : : : : : :: : : : : :: 5266 5274 : 5283 5291 : 5303 : 5316 :: 5266 5281 5291 : 5303 : 5322 5269 5291 : 5303 : 5323 5269 5292 : 5303 : 5323 5269 5297 5310 5323 5297 5310 5303 5310

   • Finding in-frame deletions

     
     

     • Find in frame deletions in INSERT.

       
       The program uses two loops corresponding to the search of the two sites in the DNA fragment to be excized from the INSERT plasmid . In this case the program must keep the translation frame after ligation. Detection of stop- codons and search for post-ligation digestions are also performed in this part of the program.
       

       CloneIt has found an in-frame deletion: Digest INSERT with Bcl I [t^gatca] (1427) and PstI [CTGCA^G] (3611). 5' --TG.TAT./GAT.C AG.GTT.CTT.ACT.G-- --CG.ACC. TGC.A/GG.CAT.GCA.AGC.T-- 3' 3' --AC.ATA. CTA.G/TC.CAA.GAA.TGA.C-- --GC.TGG./ACG.T CC.GTA.CGT.TCG.A-- 5' NH2 Y D Q V L T E -- -- T C R H A S F .COOH Treat with T4 DNA polymerase. Cloning boxes boundaries :[880-1155] [3359-3634]. Original: 5' ================================================ 3' Deletion: 5' =========......................................= 3' Equivalent to a deletion of 728 amino acids [79 %] Digestion post-ligation: BamHI Sal I Acc I Nsi I . The first stop codon detected AFTER the PstI site (3611) is localized at position 3639 on insert. Translated truncated sequence:... NSSSVPGAIKGSMAYRKRGARREANINNNDRMQEKDDEKQDQNNRMQLSDKVLSKKEEVV TDSQEEIKIADEVKKSTKEESKQLEVLKTKEEHQKEIQYEILQKTIPTFEPKESILKKLE DIKPEQAKKQTKLFRIFEPRQLPIYRANGEKELRNRTYTKLKKDTLPGDYDVREYFLNLY DR---------------------------------------------------------- ------------------------------------------------------------ ------------------------------------------------------------ ------------------------------------------------------------ ------------------------------------------------------------ ------------------------------------------------------------ ------------------------------------------------------------ ------------------------------------------------------------ ------------------------------------------------------------ ------------------------------------------------------------ ------------------------------------------------------------ ------------------------------------------------------------ --HASFCS... (N)ext (P)revious (D)iscard (S)ave sequence (A)bort (P)attern (I)nformations Te(X)t (H)TML

       See also this link

     • Look for Carboxy terminal deletions .

       
       CloneIt also proposes sites that do not necessarily create in-frame deletion but who can be also used to make C terminal deletions. In such cases, the program will propose a single site in the insert and the user will have to choose any unique site that is localized at the 3' side of the insert. Such C-terminal deletions do not have to be in-framed because they just need the presence of a stop codon after the ligation.

       CloneIt has found a Carboxy terminal deletion: Those sites that do not necesseraly create in frame deletion but they can be us ed to make Carboxy-terminal deletions. Digest INSERT with AccI [GT^MKAC] (1445). 5' --.ATC.AGT.//AT A.CAC.ATA.AAT.GAT-- 3' 3' --.TAG.TCA. TA//T.GTG.TAT.TTA.CTA-- 5' NH2 I S I H I N D .COOH Polymerase treatment may be needed in function of the second enzyme. BEWARE: Check if there is a STOP CODON after ligation. Cloning box boundaries :[641-809] [2161-2329]. Original: 5' ================================================== 3' Deletion: 5' =======================........................... 3' Equivalent to a deletion of about 294 amino acids [52 %] ´ Second Enzyme(s) that do not need polymerase modification: AccI (1445) . ´ Second Enzyme(s) that need polymerase modification: AatII (2082) . (N)ext (P)revious (D)iscard (A)bort (P)attern (I)nformations Te(X)t (H)TML

     • Find frameshifts in INSERT.

       
       A restriction enzyme can free a 5' or a 3' overhang. After digestion, fill-in and ligation on an unique site, a few bases are added or deleted from the original sequence. Thus, the frame will be shifted and the translated protein will contain one or more amino acids. Therefore, the program, uses a single computer loop corresponding to the site inside the INSERT plasmid, calculate for each site what will be the sequence modification and if a stop codon is created after the ligation.

       CloneIt has found an Enzyme that could induce frameshift. Digest INSERT with AccI [gt^mkac] (1659). 5' --.ATC.AGT.//AT A.CAC.ATA.AAT.GAT-- 3' 3' --.TAG.TCA. TA//T.GTG.TAT.TTA.CTA-- 5' NH2 I S I H I N D .COOH Treat with Klenow DNA polymerase. Beware : AccI [1 partial site] . After digestion, fill-in and ligation: 5' .ATC.AGT.ATA.TAC.ACA.TAA.ATG 3' 3' .TAG.TCA.TAT.ATG.TGT.ATT.TAC 5' NH2 I S I Y T * M COOH ¥ FrameShift (+ 2) ¥ 2 bases Added. ¥ Site is NOT reconstitued after ligation. ¥ [51 %] percentage of Insert. FIGURE: ========================= | (+2) =========================== Translated sequence:...EFELGTRGSMATFKDACYHYKRLNKLNSLVLKLGANDETRPAPMTKYKGTCLDCCQY TNLTYCRGCALYHVCQTCSQYNRCFLDEEPHLLRMRTFKDVVTKEDIEGLLTMYETLFPINEKLVNKFINSVKQRKCRNE YLLETYNHLLMPITLQALTINLEDNVYYIFGYYDCMEHENQTPFQFINLLEKYDKLLLDDRNFHRMSHLPVILQQEYALR YFSKSRFLSKGKKRLSRSDFSDNLMEDRHSPTSLMQVVRNCISiyT*... (N)ext (P)revious (D)iscard (S)ave sequence (A)bort (P)attern (I)nformations Te(X)t (H)TML

   • Minimum /Maximum percentage of insert

     
     Many of the deletions found could indeed involve sites inside the INSERT cloning boxes, while they are supposed to contain many restriction sites: the INSERT fragment would be hardly or totally deleted. Nevertheless CloneIt offers the ability to set the deletion percentage with regard to the insert length.
     Use those fields if you do not wants deletions superior to 80% of the INSERT fragment no deletions inferior to 20% of the INSERT fragment.

   • Open a REBASE File

     
     REBASE File (©New England BioLabs) is a database of all known Restriction Enzymes. It has been created by Dr. Richard J.Roberts and is freely available on ftp servor: ftp://www.neb.com/pub/rebase/ or at http://www.neb.com/rebase/link_ftpdir/. Do use an 'allenz' file to get all known enzymes.A REBASE file is a text file (ASCII)that must be present in the same folder than CloneIt.Input is case sensitive.
     

     3 NEW NON OFFICIAL FIELDS IN REBASE

     
     In order to manage temperature anb buffers I decided to add 3 new fieds to the rebase file. Those fieds are not compulsory and should be present between tag <1> and <8>. This won't give an official rebase file. You will have to write them yourself.
     • <U>:Tag for the optimal bUffer. Should be only 1 caracter. example:<U>2
     • <B>:Tag for percentage of efficiency of the four classical NEB'sBuffers. Should be wrote like this example:<B>75/0/100/50
     • <T>:Tag for optimal Temperature. By default, it is 37. example:<T>65
       
       Example:
       

       <1>Tth111I
       <2>
       <3>Thermus thermophilus strain 111
       <4>T. Oshima
       <5>GACN^NNGTC
       <U>1
       <B>100/25/25/100
       <T>65
       <6>
       <7>ADIKNQR
       <8>313,1132
       

     CloneIt use the standard symbolic abreviations.
     CloneIt doesn't handle enzymes cuting on both sides of the site ex: Bsp24I (8/13)GACNNNNNNTGG(12/7).As the REBASE file contains information about the companies selling the enzymes, the less available isoschyzomers will be discarded.

     XmaIII and Eco52I are identic [C^GGCCG]: Keep Eco52I and discard the other. XmaCI and XmaI are identic [C^CCGGG]: Keep XmaI and discard the other. Zsp2I and NsiI are identic [ATGCA^T]: Keep NsiI and discard the other. (...) Discard AccII because it is too short [CG^CG]. Discard AciI because it is too short [CCGC(-3/-1)]. Discard AciI because it is too short [CCGC(-3/-1)].

     A file is open by default at startup.
     (UNIX only) The path to this file can be declared in the system shell (ask your UNIX manager to make it for you) by the variable CLONEHOME.This was a suggestion from Dr. Gary Williams from the UK MRC Human Genome Mapping Project Ressource Centre. So you should write in your shell , for example:
     

     setenv CLONEHOME /home/virim/lindenb/ANSI/CloneIt/allenz

     
     If CLONEHOME is not declared CloneIt open the latest file used.

   • Get information about an enzyme.

     
     

     QUERY Syntax (examples)

     • Eco gaattc g^aattc
     • co G^A (1/ (
     • $6 (site length = 6)
     • _4 (all the overhangs of 4 bases)
     • _B (all the blunt sites)
     • $5_B (site length = 6 and blunt)
     • _+ (all the 3'overhanged sites)
     • _- (all the 5'overhanged sites)
     • _-4 (all the 3'overhangs 4 bases)
     • _-4|gaa (all the 3'overhangs of 4 bases and containing 'gaa')
     • $6_-4|gaa (site length=6, 3'overhangs of 4 bases and containing 'gaa')
     See also this link.

   • Enzymes Database Convertion...

     
     
     • DNA Strider Relibrary to REBASE
       It convert a DNA Strider Relibrary to a REBASE file. This allow you to use your own old Enzymes database.

       DNA Strider Relibrary	REBASE File
       Aat II, gacgt/c,	<1>AatII <5>GACGT^C <7>?

     • REBASE to DNA Strider ReLibrary
       It convert a REBASE file to a DNA Strider Relibrary . This allow you to update your DNA Srider RELibrary.

       REBASE File	DNA Strider Relibrary
       <1>AatII <2> <3>Acetobacter aceti <4>IFO 3281 <5>GACGT^C <6> <7>ADEFKLMNOPRS <8>1168,1364 <1>Bsp50I <2>FnuDII <3>Bacillus species RFL50 <4>A.A. Janulaitis <5>CG^CG <6> <7> <8>215,441,463	Aat II, gacgt/c, not available

   • Display messages.

     
     Show or hide messages

   • Speed optimization (Discard short sites) .

     
     The user can choose to discard enzymes recognizing a short sequence ( such as AluI [AG/CT] ). Those enzymes have a high probability to cleave many times and everywhere in a sequence and are insightly unuseful for cloning.

   • Use Klenow or T4 DNA Pol.

     
     The Klenow fragment and the T4 DNA polymerase are both modifying DNA polymerases whose use can be controlled by user. The Klenow and the T4 DNA polymerase possess a 5'-3' polymerase activity which synthetises a complementary DNA strand. The second polymerase has already got a strong 3'-5' exonuclease activity considerably more active on single stranded DNA than on double stranded DNA, this activity is blocked on double-stranded DNA by 5'-3' polymerase activity. Those enzymes are used to fill-in recessed ends and to create blunt extremities.
     Polymerases proprieties.
     Klenow Polymerase: 5'->3' pol.

     ..G       -----\    ..GAATT
     ..CTTAA   -----/    ..CTTAA
     

     T4 DNA Polymerase: 5'->3' pol and 3'->5' exo.
     

     ..CTGCA   -----\    ..C
     
     ..C       -----/    ..C
     

   • Number of partial digestion allowed.

     
     The user may allow up to 2 partial digestions in the strategies. Partial digestions are sites cut by the enzymes used in a cloning strategy but localized out of the VECTOR cloning box, or within the INSERT fragment. See also this link.

     • Allow partial digestion only if enzyme blunt.

       
       You can choose to allow partial digestions only if the used enzyme cuts BLUNT. Such sites won't modify the sequence if a modifying polymerase is used in a cloning strategy.

   • Continue to search a solution within a couple.

     
     For a couple of Enzyme, if CloneIt find a solution to a cloning problem there can be another solutions (Using CIP, modifying Polymerase...) that user could prefer. Nevertheless, the default solution is the easier one.

   • Allow non-overlapping overhangs.

     
     If partial overhang ligation is allowed, then such a ligation is possible:

       ..GGGT     CGGA..  --\  ..GGGTCGGA  --\  ..GGGTCGGA
       ..CCCAG       T..  --/  ..CCCAG  T  --/  ..CCCAGggT
     

   • Intersections

     
     The user is able to compare the number of sites created by each enzyme in each sequence and inside or outside each cloning box.

     _______________________________ | IN | OUT | IN | OUT | _______________________________ | VECTOR | INSERT | ___________________________________________________________ Van91I [CCANNNN^NTGG ] | 0 | 0 | 0 | 0 | VspI [AT^TAAT ] | 1 | 0 | 2 | 3 | XbaI [T^CTAGA ] | 0 | 0 | 1 | 0 | XcmI [CCANNNNN^NNNNT] | 1 | 2 | 0 | 0 | XhoI [C^TCGAG ] | 0 | 0 | 0 | 0 | XhoII [R^GATCY ] | 0 | 0 | 2 | 6 | XmaI [C^CCGGG ] | 1 | 0 | 1 | 0 | XmnI [GAANN^NNTTC ] | 0 | 2 | 1 | 1 |* ___________________________________________________________ | IN | OUT | IN | OUT | _______________________________ | VECTOR | INSERT | _______________________________ ¥: Enzyme useful for digestion post-ligation. *: Enzyme useful to determine the INSERT direction.

   • C.I.P.

     
     The usage of calf intestine alkaline phosphatase (CIAP) can be moderated. This enzyme removes the phosphate group at the 5' terminus of DNA strands, thus preventing self-annealing from the VECTOR prior to INSERT fragment insertion. If CIAP is allowed in a strategy, the program looks for enzymes cutting one time inside the INSERT fragment, one time in the VECTOR sequence that could be used to determine the fragment orientation.

     
     
     ..G      p-AATTC..  --\  ..G      AATTC..
     ..CAATT-p      G..  --/  ..CAATT      G..
     

     
     ---> Self ligation impossible.

   • Clone in frame at 5' of insert (NH2) / in frame at 3' of insert (COOH).

     
     The program can be limited to find cloning strategies that compulse the INSERT fragment to be in-frame ligated with the 5' or/and 3' VECTOR sequence surrounding ends. CloneIt searches the localization of the closest stop-codons to the cloning sites and for stop-codons created by this ligation. For example, such ligations are useful with the plasmids of the yeast two-hybrid system where the studied proteins must be in-frame synthetized with the GAL4 transcription factor domains.

   • Find strategies to clone insert into vector.

     
     The algorithm looks for any enzyme that could be used in a cloning strategy. This raw method uses four computer loops scanning each cutting site memorized. Each loop respectively corresponds to the site used to cleave the VECTOR on side 5', to the site used to cut the INSERT on side 5', to the site in the cloning boxes used to cleave the VECTOR on side 3' and to the site used to cut the INSERT on side 3'. Note that both sites used in the VECTOR can correspond to the very same site in the sequence ( linearisation of the plasmid ). Each time the site is checked in order that its localization is coherent with the position of the other one, it is also checked according to its presence in the cloning boxes boundaries and according to the number of the partial sites generated by the enzyme. Then, four possibilities are explorated whether the ligation needs or not the use of modifying polymerase, and this for both sides of the ligation . If the strategy is worth trying, it is displayed on screen. For a couple of enzymes cutting both at 3' or 5' overhang, CloneIt checks if the overhangs are complementary. Then the program is able to associate the hangs generated by a digestion by SalI[G/TCGAC] and XhoI[C/TCGAG].
     

     CloneIt V1.0 has found a solution: Digest VECTOR with EcoRI [G^AATTC] (878) and Sal I [g^tcgac] (894). 5' --G.CCG.G/AA.TT C.CCG.GGG.ATC.CG/T.CGA. CCT.GCA.GCC.AAG-- 3' 3' --C.GGC.C TT.AA/G.GGC.CCC.TAG.GC A.GCT./GGA.CGT.CGG.TTC-- 5' NH2 P E F P G I R R P A A K .COOH Digest first with EcoRI .Then treat with Klenow DNA polymerase.Finally digest with Sal I . Digest INSERT with Sca I [AGT^ACT] (1034) and Sal I [g^tcgac] (3605). 5' --AT.AAA.GT/ A.CTT.TCA.AAG.AAA.G-- --TA.GAG./TCG.A CC.TGC.AGG.CAT.G-- 3' 3' --TA.TTT.CA/ T.GAA.AGT.TTC.TTT.C-- --AT.CTC. AGC.T/GG.ACG.TCC.GTA.C-- 3' NH2 K V L S K K E -- -- E S T C R H A .COOH Treat with T4 DNA polymerase. Sites wil be in frame ligated in 5'. The first stop codon detected BEFORE the EcoRI site (878) is localized at position 428 on vector. The first stop codon detected AFTER the Sca I site (1034) is localized at position 3558 on insert. Digestion post-ligation: no enzyme was found. (N)ext (P)revious (D)iscard (N)ext solution (S)ave sequence (A)bort Te(X)t (H)TML (P)attern (I)nformations >>

   • Digestion post-ligation.

     
     The program looks for enzymes digesting the ligation mixture in order to reduce the number of non-recombinant plasmids that could still be present in the medium.

   • Saving sequences

     
     Except for the C terminal deletions, the matching sequences after ligation can be saved as a FASTA text file.

   • INSERT Pattern
     --------------
     SmaI [CCC^GGG] (897) and Bst1107I [GTA^TAC] (1659) p5_1 digestion.
      1 3890 pb      Bst1107I  1744 - SmaI  897
      2 762 pb       SmaI  897 - Bst1107I  1659
      3 85 pb        Bst1107I  1659 - Bst1107I  1744
     ***************************************************
     * Beware this strategy needs partial digestion(s) *
     ***************************************************
     

   • Information

     
     Information about enzymes extracted from the REBASE database can be displayed on screen.

     Get information about: ----------------------- 1 SmaI [CCC^GGG] 2 Bst1107I [GTA^TAC] Your choice ? /*1 < 1 < 2*/:2 -------------------------------------------------------------------------------- Information about Bst1107I [GTA^TAC]. Internet WWW link: Bst1107 I ................................................................................ INSERT Pattern -------------- Bst1107I [GTA^TAC] (1659) p5_1 digestion. 1 4652 pb Bst1107I 1744 - Bst1107I 1659 2 85 pb Bst1107I 1659 - Bst1107I 1744 ................................................................................ Prototype :SnaI Microorganism :Bacillus stearothermophilus RFL1107 Source :A.A. Janulaitis Methylation site : Commercial availability : Angewandte Gentechnologie Systeme Fermentas AB Takara Shuzo Co. Ltd. Boehringer-Mannheim New England Biolabs Refs :457 ................................................................................ Looking for Isoschizomers. (*):Commercialy available) ( ) BspM90I GTA^TAC. (*) BssNAI GTA^TAC. also available at: SibEnzyme Ltd. ( ) BstBSI GTA^TAC. (*) BstZ17I GTA^TAC. also available at: New England BioLabs ( ) XcaI GTA^TAC. --------------------------------------------------------------------------------

   • Quit.

     
     Quit the application

   • Logic Choice

     
     The most logical choice is selected by default.

   • Set sequence to Antiparalelle

     
     The sequence is set to antiparallele. This is usefull as a protein sequence should be oriented from NH2 to COOH.

   • View or create sequence

     
     If available the PICO program is used to view or create a sequence.

   • Update Rebase

     
     If available the LYNX www browser is used to jump to the rebase WWW site where you should dowload the allenz* file.

   • Saving thread

     
     All the strategies can be saved as an ASCII-text or an HTML formated file

   • Alignments

     
     (UNIX only) If available, (GCGCOREROOT defined) CloneIt can use the Gap and the BestFitprograms from the GCG Wisconsin Package to align the two DNA (or translated) sequences.

   • View Rebase

     
     If available the PICO program is used to view the rebase file.

   • Finding silent mutation

     
     After you choose the position of the amino acid you want to change, CloneIt searches for restriction enzymes that could be inserted in the sequence while providing a silent mutation.

   • Temperature and Buffer

     
     CloneIt manages the optimal temperature and the optimal buffers as it reads them from the new rebase fieds <U>, <B> and <T>.

   • Command-line

     
     

     • Command Line Memento

       
       

       -HELP  help
       
       -U	run a project -UMy_Project
       
       -VE  load vector sequence -VEpGBT9
       -VL  vector cloning box left boundary -VL855
       -VR  vector cloning box right boundary -VR900
       -VA  vector ATG position -VA644
       -VM  vector restriction map
       -IN  load insert sequence -INpbs_myGene
       -IL  insert cloning box left boundary -IL154
       -IR  insert cloning box right boundary -IR2001
       -ILi insert cloning box left internal boundary -ILi160
       -IRi insert cloning box right internalboundary -IRi1980
       -IA  insert ATG position -IA155
       -IM  insert restriction map
       -R   open a REBASE file -Rallenz
       -Y   don't use memory optimization default:FALSE
       -E   allow non-overlapping overhangs default:FALSE
       -P   don't allow use of Klenow or T4 DNA Pol default:allow them
       -T   don't allow partial digestions default:allow it
       -Z   don't allow C.I.P. default:allow C.I.P.
       -BU   don't allow incompatible buffers. default:allow incompatible buffers.
       -TP  don't allow incompatible Temperatures. default:allow incompatible Temperatures
       -N   intersections
       -GN  Gap(© GCG Wisconsin package) 
       with DNA sequences (UNIX only)
       -GP  Gap(© GCG Wisconsin package) 
       with translated sequences (UNIX only)
       -BN  BestFit(© GCG Wisconsin package) 
       with DNA sequences (UNIX only)
       -BP  BestFit(© GCG Wisconsin package) 
       with translated sequences (UNIX only)
       
       
       Cloning an INSERT into a VECTOR.
         CloneIt will try to find the best cloning strategy. It will stop when cloning conditions will be found.
       
       -A   find Cloning strategies
       -H   clone in frame at 5' of insert (NH2)  default:FALSE
       -O   clone in frame at 3' of insert (COOH) default:FALSE
       
       Finding in-frame deletions and frameshifts.
         CloneIt will try to find a maximum of cloning strategies.
       -F   find frameshifts
       -D   find in-frame deletions
       -M   Maximum percentage of insert -M80
       -m   minimum percentage of insert -m20
       -P   don't allow use of Klenow or T4 DNA Pol default:allow them
       -T   don't allow partial digestions default:allow it
       

     • Examples:

       • I wants to clone the insert of pbs_RF2 into pGBT9 in frame in 5' (NH2) using the Rebase file 'allenz'.
         ---> arguments are: -VEpGBT9 -INpbs_RF2 -Rallenz -A -H
       • Now, the frame of interest is defined on INSERT at position 430. The cloning box of pGBT9 is defined between 500 and 523.
         I don't wants to use memory optimization, C.I.P.or partial digestion.
         ---> arguments are: -VEpGBT9 -INpbs_RF2 -Rallenz -A -H -IA430 -VL500 -VR523 -Y -Z -T

   • Run a project.

     
     CloneIt can be scripted to manage large cloning projects. Here is an example of a script used to clone the insert 5 plasmids into a vector choosen by the user. It is written as an ASCII text file, located in the same folder than the application:

     ask.vector set.nh2.true set.partial.false set.pol.true set.memory.true open.rebase allenz open.insert pbs_RF2 set.vector.atg 483 cloneit open.insert pGBT9_NSP1 cloneit open.insert pGBT9_NSP2 cloneit open.insert pGBT9_NSP3 cloneit open.insert pGBT9_NSP5 cloneit

     PROJECT SYNTAX

     
     For more information, the usage of the project is comparable to the command-line.
     

     open.vector

     open the vector named at the next line

     ask.vector

     ask the user for the name of the vector

     set.vector.atg

     the atg position of the vector is set to the value of the number on the next line

     set.vector.min

     the 5' cloning box position of the vector is set to the value of the number on the next line

     set.vector.max

     the 3' cloning box position of the vector is set to the value of the number on the next line

     open.insert

     open the insert named at the next line

     ask.insert

     ask the user for the name of the insert

     set.insert.atg

     the atg position of the insert is set to the value of the number on the next line

     set.insert.min

     the 5' cloning box position of the insert is set to the value of the number on the next line

     set.insert.max

     the 3' cloning box position of the insert is set to the value of the number on the next line

     set.insert.min.int

     the 5' internal cloning box position of the insert is set to the value of the number on the next line

     set.insert.max.int

     the 3' internal cloning box position of the insert is set to the value of the number on the next line

     open.rebase

     open the rebase file named at the next line

     ask.rebase

     ask the user for the name of the rebase file

     align.Gap.dna

     align vector and insert DNA sequences with GCG's Gap© (UNIX only)

     align. Gap.protein

     align vector and insert translated sequences with GCG's Gap© (UNIX only)

     align.bestfit.dna

     align vector and insert DNA sequences with GCG's bestfit© (UNIX only)

     align.bestfit.protein

     align vector and insert translated sequences with GCG's bestfit© (UNIX only)

     set.cip.true

     CIP can be used

     set.cip.false

     do not use CIP

     set.cooh.true

     clone in frame at 3'

     set.cooh.false

     in-frame 3' cloning is not compulsory

     set.nh2.true

     clone in frame at 5'

     set.nh2.false

     in-frame 5' cloning is not compulsory

     set.partial.true

     do not use partial digestion

     set.partial.false

     partial digestions can be used

     set.pol.true

     do not use modifying polymerases

     set.pol.false

     modifying polymerases can be used

     set.memory.true

     discard short sites

     set.memory.false

     don't discard short sites

     set.part.over.true

     ligation of partial overhang can be used

     set.part.over.false

     do not allow ligation of partial overhang

     set.carboxy.true

     search for carboxy terminal deletions

     set.carboxy.false

     do not search for carboxy terminal deletions

     set.temperature.true

     can allow incompatible temperatures

     set.temperature.false

     do not allow incompatible temperatures

     set.buffer.true

     can allow incompatible buffers

     set.buffer.false

     do not allow incompatible buffers

     set.pctmin

     the minimum percentage of deletion insert is set to the value of the number on the next line

     set.pctmax

     the maximum percentage of deletion insert is set to the value of the number on the next line

     cloneit

     search for cloning strategies

     frameshift

     search for frameshift

     deltaframe

     search for in-frame deletions

   

5. Quick start

   
   

   Let's clone the Insert of plasmid "pbs-X" in-frame into the yeast "two-hybrid system" vector "pGBT9".

   • Open "pGBT9" [?]
   • You can display its sequence [?]
   • If you're not satisfied by the cloning box boundaries, do change them [?]
   • If you're not satisfied by the frame localization, do change it [?]
   • Now, open the DNA sequence containing the fragment that you wants to clone into the polylinker of pGBT9. Open pBS-X" [?]
   • If you're not satisfied by the cloning box boundaries, do change them [?]
   • If you're not satisfied by the frame localization, do change it [?]
   • Now, open the Rebase List. [?]
   • The protein coded in the fragment of pBS-X must be in-frame cloned (NH2) into the vector pGBT9: [?]
   • Select the correct menu if you don't wants to use any partial digestion [?]
   • Select the correct menu if you don't wants to use modifying polymerase [?]
   • Select the correct menu if you don't wan't to use a dephosphorylase [?]
   • NOW, SEARCH CLONING STRATEGIES[?]

   Now, let's find in-frame deletions or frameshifts into plasmid pBS-X.

   • You do not wants deletions superior to 80% of the INSERT fragment. [?]
   • You do not wants deletions inferior to 20% of the INSERT fragment. [?]
   • Now, search for in-frame deletions [?]

   Now, search for frameshifts.

   • Find frameshifts in INSERT[?]

   This is a link to the main menu.

   

6. Bibliography

   
   
   • Bartel, P., Chien, C. T., Sternglanz, R. & Fields, S. (1993). Elimination of false positives that arise in using the two-hybrid system. Biotechniques 14, 920-4.
   • Fields, S. & Song, O. (1989). A novel genetic system to detect protein-protein interactions. Nature 340, 245-6.
   • Marck, C. (1988). 'DNA Strider': a 'C' program for the fast analysis of DNA and sequences on the Apple Macintosh family of computers. Nuc leic Acids Res 16, 1829-36.
   • Nolandt, O., Kern, V., Muller, H., Pfaff, E., Theilmann, L., Welker, R. & KrŠusslich, H. (1997). Analysis of hepatitis C virus core protein interaction domains. J. Gen. Virol. 78, 1331-1340.
   • Poncet, D., Lindenbaum, P., Lharidon, R. & Cohen, J.(1997). In vivo and in vitro phosphorylation of rotavirus NSP5 correlates with its localization in viroplasms. J Virol 71, 34-41.
   • Roberts, R. J. & Macelis, D. (1998). REBASE--restriction enzymes and methylases. Nucl. Ac. Res. 26, 338-350.

   

7. Acknowledgments.

   I would like to thank Audrey Nepveu-de-Villemarceau, Janine L., C. Caron, Dr Christian Marck , Dr S. Hazout and his team, Philippe Bessieres, Christine Young, Mlle Derat, Dr Suzana Lopez (my manager ! ;-), Maria Piron ,Emmanuelle FORLOT (Je me remets au dessin demain...),Dr. Gary Williams , and Chris Boyd at MRC Human Genetics Unit for their help and their suggestions.

Pierre LINDENBAUM.
Laboratoire de Biologie Moleculaire des Rotavirus.
Virologie et Immunologie Moleculaires.
Institut National de la Recherche Agronomique.
78350 Jouy-en-Josas Cedex FRANCE.