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CloneIt: finding cloning strategies, in-frame deletions and frameshifts.

CloneIt: finding cloning strategies, in-frame deletions and frameshifts. %"  $% BIOINFORMATICS CloneIt : finding cloning strategies, in-frame deletions and frameshifts !'' !$$*# %')%!'  !%"%! %".*"!' ( %)+!'*( !'%"%! ) ##*$%"%! %".*"!'( $()!)*) )!%$"  "  '  '%$%#!&*   %*-/$/%(( , '$ Abstract Motivation: The CloneIt program searches for sub-cloning strategies, in-frame deletions and frameshifts within a plasmid sequence. Availability: The program, written in ANSI-C language, is available at http://locus.jouy.inra.fr/soft/cloneit/cloneit.html Contact: lindenb@biotec.jouy.inra.fr Molecular biologists often have to sub-clone plasmidic vectors: a DNA plasmid is cleaved and ligated with an ex- ogen DNA fragment previously excised from another plas- mid. The necessary cuts are made by restriction enzymes Fig. 1. A cloning strategy window: CloneIt displays the main which must then be carefully chosen in order to minimize the characteristics of the sub-cloning strategy. steps required to obtain the desired molecule. During the selection of those enzymes, the main difficulties encountered are due to the lack of knowledge of the enzymes’ characteris- frame to be used on each plasmid. CloneIt takes a REBASE tics, the location of the cuts within the sequence, the comple- file as the input restriction enzyme database (Roberts and mentarity between the ends, the possible self-ligation of the Macelis, 1998). vector, the orientation of the insert, the use of modifying The algorithm looks for any enzyme that could be used in DNA polymerases that generate blunt ends, the necessity to a cloning strategy. Each memorized site is checked to see clone the insert in-frame with a vector sequence, the use of whether its location is coherent with the position of another partial digestions and the creation of a stop codon after the one, its presence in the polylinker boundaries is also ligation. This exercise is very time consuming even with the checked, as well as the number of partial sites generated by help of classic DNA analysis software which only helps the the enzyme. Then, the program determines whether the strat- user by localizing restriction sites that are present a few times egy needs a modifying polymerase treatment for both sides in a plasmid sequence. All combinations cannot be checked of the ligation. If the strategy is worth trying, it is displayed by the scientist, so a simple cloning strategy can be missed. on screen. Moreover, CloneIt checks whether restriction en- A program was developed to find sub-cloning strategies, zymes produce compatible cohesive ends. The program is in-frame deletions and frameshifts quickly in a plasmid se- then able to ligate the ends generated by a digestion by quence while still controlling the problems described above. SalI[G/TCGAC] and XhoI[C/TCGAG]. This program is not an expert system, as it does not ‘learn’ If plasmid dephosphorylation is allowed in a strategy, the the logical steps accomplished by the biologist and it does not program searches for enzymes that could be used to deter- have to be accompanied in its search: it simply runs an algo- mine the fragment orientation. If the ligation must be per- rithm that explores all the possible enzyme combinations that formed in order to maintain the frame, CloneIt searches for could be used to clone the molecules. the location of the closest stop codons to the cloning sites and Two sequences are used: the ‘VECTOR’ and the ‘IN- for stop codons created by this ligation. The program looks SERT’ plasmid. The ‘VECTOR’ plasmid is designed to be for enzymes digesting the ligation mixture in order to reduce the plasmid where the user wants to subclone a DNA frag- the number of non-recombinant plasmids that could still be ment excised from the ‘INSERT’. The program uses a short present in the medium (Fig. 1). database to try to localize the bounds of the polylinkers. If the In order to find in-frame deletions, the program searches coding frame is of importance, the program can find the best for the two sites in the DNA fragment to be excised from the Oxford University Press 465 P.Lindenbaum INSERT plasmid while keeping the translation frame. Clo- displayed on screen. Ultimately, a simple scripting language neIt also proposes sites that do not necessarily create in- can be used in order to manage large cloning projects. frame deletions, but that can be used to make carboxy-ter- minal deletions which do not have to keep the translation Acknowledgements frame. I would like to thank A.Nepveu-de-Villemarceau, M.Piron, CloneIt can find sites producing frameshifts. A restriction J.Lindenbaum, S.Hazout and his team, and C.Young for their enzyme can free a 5′ or a 3′ overhang. Then after fill-in and valuable help. ligation, 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. References The matching sequences after ligation can be saved as a text file. Moreover, the digestion pattern and information Roberts,R.J. and Macelis,D. (1998) REBASE: restriction enzymes and about enzymes extracted from the REBASE database can be methylases. Nucleic Acids Res., 26, 338–350. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioinformatics Oxford University Press

CloneIt: finding cloning strategies, in-frame deletions and frameshifts.

Lindenbaum, P
Bioinformatics , Volume 14 (5): 2 – Jun 1, 1998
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Publisher
Oxford University Press
Copyright
© Published by Oxford University Press.
ISSN
1367-4803
eISSN
1460-2059
DOI
10.1093/bioinformatics/14.5.465
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Abstract

%"  $% BIOINFORMATICS CloneIt : finding cloning strategies, in-frame deletions and frameshifts !'' !$$*# %')%!'  !%"%! %".*"!' ( %)+!'*( !'%"%! ) ##*$%"%! %".*"!'( $()!)*) )!%$"  "  '  '%$%#!&*   %*-/$/%(( , '$ Abstract Motivation: The CloneIt program searches for sub-cloning strategies, in-frame deletions and frameshifts within a plasmid sequence. Availability: The program, written in ANSI-C language, is available at http://locus.jouy.inra.fr/soft/cloneit/cloneit.html Contact: lindenb@biotec.jouy.inra.fr Molecular biologists often have to sub-clone plasmidic vectors: a DNA plasmid is cleaved and ligated with an ex- ogen DNA fragment previously excised from another plas- mid. The necessary cuts are made by restriction enzymes Fig. 1. A cloning strategy window: CloneIt displays the main which must then be carefully chosen in order to minimize the characteristics of the sub-cloning strategy. steps required to obtain the desired molecule. During the selection of those enzymes, the main difficulties encountered are due to the lack of knowledge of the enzymes’ characteris- frame to be used on each plasmid. CloneIt takes a REBASE tics, the location of the cuts within the sequence, the comple- file as the input restriction enzyme database (Roberts and mentarity between the ends, the possible self-ligation of the Macelis, 1998). vector, the orientation of the insert, the use of modifying The algorithm looks for any enzyme that could be used in DNA polymerases that generate blunt ends, the necessity to a cloning strategy. Each memorized site is checked to see clone the insert in-frame with a vector sequence, the use of whether its location is coherent with the position of another partial digestions and the creation of a stop codon after the one, its presence in the polylinker boundaries is also ligation. This exercise is very time consuming even with the checked, as well as the number of partial sites generated by help of classic DNA analysis software which only helps the the enzyme. Then, the program determines whether the strat- user by localizing restriction sites that are present a few times egy needs a modifying polymerase treatment for both sides in a plasmid sequence. All combinations cannot be checked of the ligation. If the strategy is worth trying, it is displayed by the scientist, so a simple cloning strategy can be missed. on screen. Moreover, CloneIt checks whether restriction en- A program was developed to find sub-cloning strategies, zymes produce compatible cohesive ends. The program is in-frame deletions and frameshifts quickly in a plasmid se- then able to ligate the ends generated by a digestion by quence while still controlling the problems described above. SalI[G/TCGAC] and XhoI[C/TCGAG]. This program is not an expert system, as it does not ‘learn’ If plasmid dephosphorylation is allowed in a strategy, the the logical steps accomplished by the biologist and it does not program searches for enzymes that could be used to deter- have to be accompanied in its search: it simply runs an algo- mine the fragment orientation. If the ligation must be per- rithm that explores all the possible enzyme combinations that formed in order to maintain the frame, CloneIt searches for could be used to clone the molecules. the location of the closest stop codons to the cloning sites and Two sequences are used: the ‘VECTOR’ and the ‘IN- for stop codons created by this ligation. The program looks SERT’ plasmid. The ‘VECTOR’ plasmid is designed to be for enzymes digesting the ligation mixture in order to reduce the plasmid where the user wants to subclone a DNA frag- the number of non-recombinant plasmids that could still be ment excised from the ‘INSERT’. The program uses a short present in the medium (Fig. 1). database to try to localize the bounds of the polylinkers. If the In order to find in-frame deletions, the program searches coding frame is of importance, the program can find the best for the two sites in the DNA fragment to be excised from the Oxford University Press 465 P.Lindenbaum INSERT plasmid while keeping the translation frame. Clo- displayed on screen. Ultimately, a simple scripting language neIt also proposes sites that do not necessarily create in- can be used in order to manage large cloning projects. frame deletions, but that can be used to make carboxy-ter- minal deletions which do not have to keep the translation Acknowledgements frame. I would like to thank A.Nepveu-de-Villemarceau, M.Piron, CloneIt can find sites producing frameshifts. A restriction J.Lindenbaum, S.Hazout and his team, and C.Young for their enzyme can free a 5′ or a 3′ overhang. Then after fill-in and valuable help. ligation, 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. References The matching sequences after ligation can be saved as a text file. Moreover, the digestion pattern and information Roberts,R.J. and Macelis,D. (1998) REBASE: restriction enzymes and about enzymes extracted from the REBASE database can be methylases. Nucleic Acids Res., 26, 338–350.

Journal

Bioinformatics – Oxford University Press

Published: Jun 1, 1998

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