CLONEIT
AN ANSI-C PROGRAM FINDING SUB-CLONING STRATEGIES, IN-FRAME DELETIONS AND
FRAMESHIFTS USING RESTRICTION ENZYMES AND DNA POLYMERASES.
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
made 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 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
+ 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/clonit.html
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Samples
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Restriction Map
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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
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Finding in-frame deletions
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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----------------------------------------------------------
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--HASFCS...
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Finding frameshifts in INSERT.
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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:
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| (+2)
===========================
Translated sequence:...EFELGTRGSMATFKDACYHYKRLNKLNSLVLKLGANDETRPAPMTKYKGTCL
YTNLTYCRGCALYHVCQTCSQYNRCFLDEEPHLLRMRTFKDVVTKEDIEGLLTMYETLFPINEKLVNKFINSVKQ
EYLLETYNHLLMPITLQALTINLEDNVYYIFGYYDCMEHENQTPFQFINLLEKYDKLLLDDRNFHRMSHLPVILQ
RYFSKSRFLSKGKKRLSRSDFSDNLMEDRHSPTSLMQVVRNCISiyT*...
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SubCloning
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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.
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Informations
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Get information about Bst1107 I
INSERT Pattern
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Bst1107I [GTA^TAC] (1659) p5_1 digestion.
1 4652 pb Bst1107I 1744 - Bst1107I 1659
2 85 pb Bst1107I 1659 - Bst1107I 1744
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.
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
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.
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.
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