Finding a cloning strategy ?

Pierre Lindenbaum lindenb at jouy.inra.fr
Mon Dec 21 05:39:02 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 ?


The use of plasmid vectors in cloning strategies is the basis of any
molecular biologist repertoire. Efficiency is essential and requires the
use of the most appropriate restriction enzymes choices for the desired
application. Selection of those enzymes remains a skill. Several points
that merit consideration include (1) the enzymes characteristics, (2)
location of the restriction sites within the sequence, (3) complementarity
of protuding ends, (4) possible self ligation, (5) use of modifying DNA
polymerase generating blunt ends, (6) in frame cloning constraints,(7) use
of partial digestions and (8) the creation of a stop codon at the ligation
site. This exercise is labor intensive, even with the help of the
classical DNA analysis softwares. These programs typically facilitate
localizing restriction sites in a plasmid sequence;  however it is
impossible to examine all restriction sites and /or combinations then, a
simple cloning strategy may be over-looked as a result of incomplete or
inadequate searching. 

The CloneIt program was created in order to quickly find cloning 
strategies (including sub-cloning, in-frame deletions and frameshifts) 
while controlling the problems described above.

CloneIt is available at the following URL: 

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


------------------------------------------------------------------------------
  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


..............................................................................
Intersections
..............................................................................

                            _______________________________
                            |    VECTOR    |    INSERT    |
                            _______________________________
                            |  IN  |  OUT  |  IN  |  OUT  |
___________________________________________________________
  AatII    [GACGT^C       ] |    0 |    1  |    0 |    1  |
  AccI     [GT^MKAC       ] |    1 |    3  |    1 |    3  |
  AccIII   [T^CCGGA       ] |    0 |    0  |    0 |    0  |
  Acc65I   [G^GTACC       ] |    0 |    1  |    2 |    0  |
  AccBSI   [CCGCTC(-3/-3) ] |    0 |    3  |    0 |    3  |

(...)

  VspI     [AT^TAAT       ] |    0 |    6  |    3 |    6  |
  XbaI     [T^CTAGA       ] |    0 |    0  |    2 |    1  |
  XcmI     [CCANNNNN^NNNNT] |    0 |    2  |    0 |    0  |
  XhoI     [C^TCGAG       ] |    0 |    0  |    0 |    1  |
  XhoII    [R^GATCY       ] |    2 |    7  |    2 |    6  |
  XmaI     [C^CCGGG       ] |    1 |    0  |    1 |    0  |
  XmnI     [GAANN^NNTTC   ] |    0 |    4  |    0 |    3  |
___________________________________________________________
                            |  IN  |  OUT  |  IN  |  OUT  |
                            _______________________________
                            |    VECTOR    |    INSERT    |
                            _______________________________


----------------------------------------------------------------------------
   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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