Document update: October 1998


An ANSI-C program finding sub-cloning strategies, in-frame deletions and frameshifts using restriction enzymes and DNA polymerases.

LINDENBAUM Pierre (1998) Bioinformatics/CABIOS Vol.14;5,1998 pp465-466

current version is 2.0

  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 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):

    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:
    this creates a program called "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. ___________________________________________________ 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

  5. Quick start

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

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

    Now, search for frameshifts.

    This is a link to the main menu.

  6. Bibliography

  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.

If you are interested in being kept informed directly about CloneIt , please send me your e-mail address.

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

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