A Program Finding Cloning Strategies

Pierre Lindenbaum lindenb at jouy.inra.fr
Mon Nov 2 04:29:58 EST 1998


     _________________________________________________________________

                                    CLONEIT(tm)

   AN FREE 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
     _________________________________________________________________


 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.
          + the buffers and the temperatures compatibilities.

   This exercise takes a long time even with a computorized help of the
       classic DNA analysis softwares 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.
       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
          + handle temperatures and buffers.
          + 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.
------------------------------------------------------------------------------

   CloneIt source code

        The source code is freely available at

        http://locus.jouy.inra.fr/soft/cloneit/cloneit.html


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

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.

..............................................................................
        Finding in-frame deletions
..............................................................................

 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...

..............................................................................
Finding frameshifts in INSERT.
..............................................................................

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:...EFELGTRGSMATFKDACYHYKRLNKLNSLVLKLGANDETRPAPMTKYKGTCL
YTNLTYCRGCALYHVCQTCSQYNRCFLDEEPHLLRMRTFKDVVTKEDIEGLLTMYETLFPINEKLVNKFINSVKQ
EYLLETYNHLLMPITLQALTINLEDNVYYIFGYYDCMEHENQTPFQFINLLEKYDKLLLDDRNFHRMSHLPVILQ
RYFSKSRFLSKGKKRLSRSDFSDNLMEDRHSPTSLMQVVRNCISiyT*...

..............................................................................
Informations
..............................................................................


Get information about 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.
..............................................................................
Restriction Map
..............................................................................


                                     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



----------------------------------------------------------------------------
   LINDENBAUM Pierre (1998) Bioinformatics/CABIOS Vol.14;5,1998 pp465-466
----------------------------------------------------------------------------
098)o%:::%o(8609
.6o%:%o(86098)..........................................................
  (86098)o
6098)o%::%o9        Pierre LINDENBAUM
098)o%::::::%o9      Biologie Moleculaire des Rotavirus
 6o%::::::%o(860      Unite de Virologie et Immunologie Moleculaires
    6o%::%o(8609       Institut National de la Recherche Agronomique
      o(86098)          78352 JOUY-EN-JOSAS CEDEX
  (86098)o%:%o9          FRANCE
6098)o%:::%o(860    
098)o%:::%o(8609      lindenb at biotec.jouy.inra.fr   
 6o%:%o(86098)        
  (86098)o
6098)o%::%o9............................................................
098)o%::::::%o9 
 6o%::::::%o(860
   CloneIt WWW Home Page:http://locus.jouy.inra.fr/soft/cloneit/cloneit.html




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