OligoMutantMaker (version 1.0)
Copyright 1987 Kevin Beadles, David Canter, Arnold Berk
("readme.doc" added 8/26/88, 2/24/89 by Spencer Yeh)
INTRODUCTION
OligoMutantMaker simplifies the designing and screening of
oligonucleotide-directed single amino acid substitution experiments by
searching for nucleotide sequences which introduce a restriction
endonuclease recognition sequence into the codon substitution site of the
mutant.
The program utilizes the redundancy of the genetic code to generate
all
possible nucleotide sequences for a given amino acid substitution
(including nucleotide sequences in which silent mutations are introduced
into the 5' and/or 3' codons immediately adjacent to the substitution
site)
and determines whether any restriction endonuclease recognition
sequences are present. Any nucleotide sequence containing a restriction
site is displayed/printed along with relevant information about the site
such as its restriction enzyme(s), the random frequency of the enzyme's
recognition sequence(s), the prototype of the enzyme, isoschizomers of
the
enzyme, and the unit cost of the enzyme from various biochemical
suppliers.
CONTACT ADDRESS
For questions about the program or suggestions for future improvements
please contact:
Kevin Beadles
1044 1/2 Shrader St.
San Francisco, CA 94117
tel.: (415) 759-0148
(Kevin will call you back collect if he must return your call.)
SYSTEMS SUPPORTED
An IBM compatible computer running MS-DOS is needed.
AVAILABILITY
OligoMutantMaker is available from the BIONET Lending Library. Please
send a stamped, self-addressed return envelope along with a formatted
diskette (specify capacity) with your request to:
BIONET Administrator
BIONET/ IntelliGenetics
700 East El Camino Real, Suite 300
Mountain View, CA 94040
tel.: (415) 962-7337
PROGRAM FILES before de-ARCing
README.DOC
BWMUTANT.COM
CMUTANT.COM
CUTTERS.DAT
CODONID.DAT
[ENZYME.TXT]
(BIONET diskette version)
This documentation file
Executable file for monochrome monitors
Executable file for color monitors
Binary database of enzyme cleavage and availability data
Standard genetic code
This file is created everytime the program is run.
It contains a copy of the results of the last
analysis.
PROGRAM FILES before de-ARCing
(BIONET downloadable version)
README.DOC
This documentation file.
OLIGO.UUE Archived and uuencoded file for both monochrome
and color executable versions, and associated data
files. (234 Kb).
PROGRAM FILES after de-ARCing (Approx. 350 Kb total)
README.DOC
BWMUTANT.COM
CMUTANT.COM
CUTTERS.DAT
CODONID.DAT
[ENZYME.TXT]
This documentation file
Executable file for monochrome monitors
Executable file for color monitors
Binary database of enzyme cleavage and availability data
Standard genetic code
This file is created everytime the program is run.
It contains a copy of the results of the last
analysis.
DOCUMENTATION
The program is internally documented with help messages.
STARTING THE PROGRAM
On the BIONET diskette version, the program files are not compressed,
whereas the ".uue" files in the downloadable version require both the
UUDECODE program and an "ARC"-compatible dearchiving program such as
PKUNPAK to restore the executable file.
".UUE" files:
First decode the ".uue" file:
>UUDECODE oligo.uue
Then dearchive the resulting ".arc" file to an appropriate directory by
using PKUNPAK (or compatible program):
>PKUNPAK oligo.arc c:\oligo
Once installed, CD to the appropriate directory and then start the
program by typing its name at the MS-DOS prompt:
or
>BWMUTANT
(for monochrome monitors)
>CMUTANT
(for color monitors)
It is recommended that the user copy the program files to hard disk so
that more disk space will be available to the program.
SAMPLE PROGRAM OUTPUT
The following is abreviated output from a sample run to give an idea of
how the program works:
OligoMutantMaker requires the entry of nine codons (27 nucleotides)
from the "sense"--or mRNA-homologous--strand of DNA. The codons are
are numbered "-4" through "+4" (upstream-to-downstream or 5'-to-3')
where codon #0 is the codon to be substituted. The codons must be
entered
individually using standard one letter nucleotide abbreviations (A, C, G,
T
or U) as they are requested.
(The user enters the 27 nucleotide fragment at this point)
Nucleotide #
1
4
7
Nucleotide Sqn
GGG GGG GGG GGG GGG GGG GGG GGG GGG
Codon #
-4
Amino Acid Sqn
GLY GLY GLY GLY GLY GLY GLY GLY GLY
-3
-2
10
-1
13
0
16
1
19
2
22
3
25
4
(Once the fragment is entered, the user picks an amino acid replacement
for the central codon)
1) ALA
9) HIS
17) THR
2) ARG
10) ILE
18) TRP
3) ASN
11) LEU
19) TYR
4) ASP
12) LYS
20) VAL
5) CYS
13) MET
21) STP
6) GLN
14) PHE
7) GLU
15) PRO
8) GLY
16) SER
Please enter the number of the amino acid that you want to insert
at codon #0: 14
<D>isplay
<P>rint
<R>estart
(The results are then displayed. Lowercase letters indicate altered
base pairs. In the description of the recognition sequence, the "/"'s
indicate where the enzyme cuts on each of the strands.)
GGG GGG GGG GGa ttc GGG GGG GGG GGG -- the recognition sequence is GANTC
-- --Hinf I
5' G/ANT C
3' C TNA/G
random site frequency: 1/256
isoschizomers: FnuA I, Hha II, Nca I, Nov II, NsiH I, Hine I
BMB: 4.4c/u 1000u $44
BRL: 4c/u 1000u $40
NEB: 1.1c/u 5000u $55
USBC: 3.5c/u 1000u $35
<D>isplay
<P>rint
<R>estart
ADDITIONAL INFORMATION ABOUT THE PROGRAM
Restriction Site Location:
OligoMutantMaker searches for restriction sequences which have at
least one nucleotide in codon #0 (i.e. the codon to be substituted).
Since
the longest recognition sequence (GGCCNNNNNGGCC--Sfl I) spans thirteen
nucleotides, it is necessary to enter 27 nucleotides (twelve nucleotides
on
either side of the three nucleotides which comprise codon #0) into the
program in order for it to function.
"Silent" Mutations:
The program utilizes the redundancy of the genetic code to generate
all possible nucleotide sequences for the given amino acid substitution
in
codon #0 (i.e. the codon to be substituted). In addition to searching
for
restriction sites which match these nucleotides sequences, the program
also examines those nucleotide sequences in which "silent" mutations have
been introduced into codon #-1 and codon #+1 (i.e. the codons immediately
upstream--or 5'--and downstream--or 3'--from codon #0, respectively)
and determines whether any additional restriction sites are created.
Only
those "silent" mutations which can be created by a single substitution in
the nucleotide closest to codon #0 (i.e. the third--or "wobble"-nucleotide
of codon #-1 and the first nucleotide of codon #+1) are examined.
Therefore, only five nucleotides (centered upon the middle nucleotide of
codon #0) are subject to substitution.
Abbreviations used in OligoMutantMaker:
standard three letter amino acid abbreviations
STP: stop codon (UAA, UAG, or UGA)
standard one letter nucleotide abbreviations
N: any nucleotide
m: methylated nucleotide
/: cleavage site
standard restriction endonuclease abbreviations
BMB: Boehringer Mannheim Biochemicals
BRL: Bethesda Research Laboratories
NEB: New England Biolabs
USBC: United States Biochemical Corperation
u: unit(s)
c/u: cents/unit
Miscellaneous Notes:
OligoMutantMaker recognizes 164 different restriction sites which are
are cleaved by 126 different restriction enzymes.
Price comparisons are based upon the smallest low concentration order
available from each supplier.
OligoMutantMaker does not search for cleavage specificities generated
by methylating DNA with a selected restriction endonuclease (see
NEB
1986/87 Catalog p32 & p123 for further details).
OligoMutantMaker does not note enzyme specificities resulting from
star activity (NEB 1986/87 Catalog p86 & p121).
Bibliography:
Boehringer Mannheim Biochemicals, 1987/88 (Catalog)
Bethesda Research Laboratories, Catalog & Reference Guide (1985)
New England Biolabs, Catalog 1986/87
United States Biochemical Corporation, Enzymes & Reagents for
Molecular Biology 1987