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Summary
The
sequential deletion method is generally used to locate the
functional domain of a protein. In order to find the various
N-terminal truncated mutants, researchers have to investigate the
ATG-like codons, to design various PCR forward primers and to do
several PCR experiments. This web server (N-terminal Truncated Mutants
Generator for cDNA) will automatically generate a group of forward PCR
primers and a corresponding reverse PCR primer that can be used in a
single batch of a multiplex PCR experiment to extract the various
N-terminal truncated mutants. This saves much time and money for
those who use the sequential deletion method in their research.
INPUT
1. The target cDNA sequence.
2. The addresses of the start codon and the stop codon.
3. Multiplex PCR primer design criteria including melting
temperature (Tm), primer length, GC contents, internal
self-complement, cross dimerization, terminal limitation, and
specificity to filter out unqualified forward PCR primer candidates.
PROCESSING METHOD
First, it considers the in-frame
criterion and finds ATG-like codons, ATX,AXG or XTG with X
represents A, T, G, or C, then ATG-like codons are modified to ATG start codon for
signaling the initiation of translation in a cDNA sequence.
Secondly, segments containing ATG-like codons within a window of
variable length between 18 to 24 nucleotides are selected as
candidates. By sliding the window one nucleotide at a time along the
cDNA sequence, a stack of PCR primer candidates is collected for
each ATG-like codon. The third step checks the criteria including
melting temperature (Tm), primer length, GC contents,
internal self-complement, dimerization, terminal limitation, and
specificity to filter out unqualified forward PCR primer candidates.
In the fourth step, the hierarchical cluster algorithm is used for
classifying the forward primer candidates into several groups.
Finally, the best forward primer grouping is selected and then one
or several
reverse primers meeting the above criteria and compatible with the
forward primers are chosen. The designed forward primers
accompanied with a common reverse primer can work in a single batch
of a multiplex PCR experiment that is effective in extracting
various N-terminal truncated mutants of a protein for discovery of
its functional domain.
OUTPUT
Several groups of the forward PCR primers accompanied with the
reverse PCR primer, the melting temperature, the lengths of primers
and the lengths of various mutants.
USER's GUIDE
1.Click the "PROCESS" at the bottom to begin the processing.
2.Click the "Example.0" , "Example.1" and "Example.2" will show
examples.
3.Default input values are provided but user may change the values.
Note that some input value settings (e.g. setting Primer Melting
Temperature to 45-80) may cause the server to take a much longer
time to obtain results. Also note that with some input value
settings (e.g. setting Specificity to 40), the server may find no
primers that satisfy the criterion. In this case, the server will
stop and report this fact.
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