Download insilico.mutagenesis.help.me.please

insilico.mutagenesis.help.me.please
This tutorial provides additional information to simplify the use of the insilico mutagenesis
tool. This short tutorial will guide you through the input fields of the user interface and enable
you to perform your own mutagenesis primer predictions.
Fill in the information on the main input page, necessary to calculate the mutagenesis
primers, as outlined in detail below.
1.) TARGET NUCLEOTIDE SEQUENCE:
The program asks you to provide your target nucleotide sequence (1), which includes flanking vector
sequences up-and downstream of the gene (or target region: e.g. untranslated intergenic regions like
promotors) of interest. Please provide a plain nucleotide sequence. All non-DNA characters (e.g. as in
FASTA formatted sequences) will cause an error. The addition of flanking nucleotide sequences is
only necessary if a complete saturation or sequence scan of your protein is intended. For
example if you want to substitute each and every single amino acid of your protein against a given
amino acid. In this case the addition of flanking vector sequences is necessary since the program will
design mutagenesis oligos for the first and last five codon (amino acid) exchanges by including
stretches of the vector which will be used as template in the mutagenesis PCR to amplifiy a full length
mutated product in one single PCR. Usually 40 bp up- and downstream of the target sequence are
should be used.
2.) SEQUENCE IDENTIFIER (NAME)
It is very important to choose a unique sequence identifier, which enables the program, after data
generation, to identify your mutagenesis project in our database. For a subsequent restriction analysis
as well as for outputfile generation the program will ask you to provide your chosen sequence identifier
for security reasons. Furthermore, your generated data will be stored in our database for one week.
During this period of time your results remain accessible by pressing the generate output button in the
navigation bar and providing your sequence identifier. After one week the generated data (tables and
files) will be deleted completely from the server.
3.) MUTAGENESIS CODON:
Please select a mutagenesis codon from the pull down menu. For each amino acid more than one
codon is listed depending on the redundancy of the genetic code. For optimal expression the codon
usage of your expression host should be taken into acount, when selecting the mutagenesis codon. For
a saturation mutagenesis approach select the degenerated codon "NNS" or “NNN”.
4.) TARGET REGION (ORF or untranslated intergenic region)
It is necessary to define the start and stop positions of your target region (open reading frame or
intergenic region) within the overall sequence, whereas START refers to the first base pair, ( for open
reading frames usually the “A” of the ATG start codon), and STOP to the last base pair of your target.
In case you are providing a sequence without flanking vector sequences and a mutagenesis of a certain
region within your target sequence is desired, please also provide this information.
Example: 600bp random nucleotide sequence: ORF from 45-555 corresponds to a protein of 170 amino
acids ( region highlighted in red )
45
ttgagaagttatctgggcgccgaccgcactctgtacacattcgtATGccctaaaaggtaccgtagaggctggcct
taaattcgtccgatggcaccacaatagcaggcgaccataagaagggcgtgagctctgcccatcttgaatgtgacc
gacccacaaaggtaaattcgtcatttctggaccttaagcgtaactgtatacagcacagttacattatctactcgg
gcaatatgtacatctgaatagattgtccttagcaccacagcgttccgccgagcgagttgtttatgtcttaaatgc
tacacccgtcagggcttcacagtgaattacaatctgattcttgtcctgatactctgttattttgaggtagacaaa
agcccgtgggggcagttcctacgctttgtttagggggtaaactacctctacgagtctatagcagcttgcacccga
ccaaccgacagggtggaatcttggtaggaatccctacagctatattgcacgtacaggcataggtataccgagaat
cactgtcgtggggcattaatgtgtcccTAAgtcctctcgcaccagatcacgtagaccttcgtgagcacgacatg
555
GENE REGION: Start: 45
Stop: 555
5.) REGION TO MUTATE:
Define the region you want to mutate. The program will generate primers for a subsequent codon (or
base triplett) exchange within this region. Provide the region starting from the first base pair and ending
with the last base pair.
Example: putative ORF from 45-555, mutagenesis from first codon after start codon ATG until last
codon before stop codon TAA ( region highlighted in grey, start and stop codon in red)
48
ttgagaagttatctgggcgccgaccgcactctgtacacattcgtATGccctaaaaggtaccgtagaggctggcct
taaattcgtccgatggcaccacaatagcaggcgaccataagaagggcgtgagctctgcccatcttgaatgtgacc
gacccacaaaggtaaattcgtcatttctggaccttaagcgtaactgtatacagcacagttacattatctactcgg
gcaatatgtacatctgaatagattgtccttagcaccacagcgttccgccgagcgagttgtttatgtcttaaatgc
tacacccgtcagggcttcacagtgaattacaatctgattcttgtcctgatactctgttattttgaggtagacaaa
agcccgtgggggcagttcctacgctttgtttagggggtaaactacctctacgagtctatagcagcttgcacccga
ccaaccgacagggtggaatcttggtaggaatccctacagctatattgcacgtacaggcataggtataccgagaat
cactgtcgtggggcattaatgtgtcccTAAgtcctctcgcaccagatcacgtagaccttcgtgagcacgacatg
552
REGION TO MUTATE:
first base:
48
last base: 552
6.) OLIGO SWITCH POSITION:
It has been proven practical to design primers in a way, that the megaprimers, generated in first PCR
reaction, should not exceed a certain length because megaprimers, which are too long, might cause
problems in the second PCR due to inefficient elongation or priming of those megaprimers. Usually half
the size of the gene of interest is used, e.g for the 510 bp putative ORF reverse mutagenesis primers
are designed until the specified oligo-switch position (in our example above 300 bases ) is reached.
From this position until the end of the region to mutate forward mutagenesis primers are designed.
Therefore using these mutagenesis primers together with a respective vector specific forward or reverse
primer, Megaprimers up to an size of app.255 bp. are generated during the first PCR reactions.
However, if only a short region of your gene has to be mutated it is not necessary to provide an oligoswitch position. In this case this field should be ignored.
Example: 501 bp gene corresponds to an 167 amino acid protein. Oligo-Switch Position( highlighted in
blue ): 250 bp downstream of the start codon (position 295 within the 600 bp sequence (gene+ flanking
vector parts)
48
ttgagaagttatctgggcgccgaccgcactctgtacacattcgtATGccctaaaaggtaccgtagaggctggcct
taaattcgtccgatggcaccacaatagcaggcgaccataagaagggcgtgagctctgcccatcttgaatgtgacc
gacccacaaaggtaaattcgtcatttctggaccttaagcgtaactgtatacagcacagttacattatctactcgg
295
gcaatatgtacatctgaatagattgtccttagcaccacagcgttccgccgagcgagttgtttatgtcttaaatgc
tacacccgtcagggcttcacagtgaattacaatctgattcttgtcctgatactctgttattttgaggtagacaaa
agcccgtgggggcagttcctacgctttgtttagggggtaaactacctctacgagtctatagcagcttgcacccga
ccaaccgacagggtggaatcttggtaggaatccctacagctatattgcacgtacaggcataggtataccgagaat
cactgtcgtggggcattaatgtgtcccTAAgtcctctcgcaccagatcacgtagaccttcgtgagcacgacatg
552
Now press the "do.mutagenesis" button. A new window will be
opened containing a table with the generated primer information
(primer name, sequence, length and thermodynamic melting
temperature).
When following the link to the restriction analysis tool, which can be found on the previous
output page, a new window will appear asking you to provide additional information to
perform a restriction analysis:
Usually two restriction enzymes are used for subsequent cloning of the full-length mutated PCR product.
Recognition sites for such enzymes should not be present within the target of interest, despite at the 5’and 3’- ends to enable sticky or blunt-end cloning. This tool allows you to search for such recognition
sites which might interfere with the desired cloning strategy.
Please provide the same sequence identifier as on the first input-window. Only the correct sequence
identifier enables the program to identify your sequence in the database. Additionally, the names of two
restriction enzymes have to be provided, which should not be present in the target gene after
mutagenesis.
Please note that for a saturation mutagenesis approach, in which the degenerate
"NNS" or “NNN” codon is introduced into the oligos a restriction analysis is not
possible and should carried out.
Now press the "analyze" button. A new window will be opened,
containing the number of restriction enzyme recognition sites
for the two provided enzymes per generated oligo. In case a
new, unwanted, recognition site is generated which interferes
with the desired cloning strategy, the respective primer has to
be redesigned manually.
To generate output-files containing the primer sequences (as
please
press
the
Microsoft
ExcelTM-Spreadsheet)
"generate.output" button on the navigation bar and follow the
respective links to download your results.
For additional help, read the help pages at www.insilico.uni-duesseldorf.de or send an email
to the author of the insilico.mutagenesis tool.