Protein Protocols Protein Protocols

Sequence Analysis with WinGene/WinPep 743
3.2. WinGene
3.2.1. Miscellaneous
With WinGene, nucleotide sequences can be easily reverse-complemented by clicking
on the “Complement the Sequence” button. After selecting part of the sequence
the “Primer check” function calculates base composition and melting temperature of
the selected oligonucleotides (3). If there was no selection the dialog will be opened for
de novo entry of the oligo-sequence. The resulting report can be printed or pasted into
any text processor software.
3.2.2. Restriction Analysis
Starting with version 2.0, WinGene performs restriction analysis of a given
sequence. The source file for restriction enzymes is gcg.009 from the REBASE website
(2) (see Note 2). In the “Restriction Analysis Dialog,” the options for the current task
can be set. The user can choose one of the following enzyme subgroups: (1) all
enzymes, (2) only enzymes with recognition sites equal to or greater than a defined
value, (3) a prechosen, saved subset of enzymes (YFE, your favorite enzymes), or (4) a
chosen subset out of the list of all enzymes. To generate a YFE list: Select all desired
enzymes into the right list box. Then, click on “Make YFE.” The file YFE01.txt will be
created in the program directory. This file contains the ID numbers of the selected
favorite enzymes for fast and convenient access with subsequent analysis (see Note 3).
In addition, the selected subset can be restricted further by activating “Only commercially
available enzymes.” The provided sequence will be treated as linear unless the
“Circular DNA” item is checked.
Output may contain the sequence with indicated restriction sites (“Map”) and/or a
table of all occurrences of the recognition sequences in the input sequence (“Table”).
Recognition sites can be marked automatically by bold letters if desired.
3.2.3. Translation and Identification of Open Reading Frames
Translation of the nucleotide sequence is achieved by clicking on the “Translate the
Sequence” button. The translate window displays the sequence with its translation in
three frames below. Basic text formatting (bold, underlined, italic) is available.
WinGene will indicate which sequence (original or complementary) is used, that is,
corresponds to the upper strand. The individual strands and frames can be deselected.
This feature is useful if display of only the upper strand with one translated frame is
desired. A list of all ORFs longer than the preset minimum (see Note 4) is available
with the position of the first occurring Met given. If only one frame is displayed the
longest ORF in this frame can be exported directly into WinPep (“Export into WinPep”
in the file menu).
3.3. WinPep
3.3.1. Physicochemical Properties
A basic set of calculations determines some physicochemical properties of the given
peptide, including amino acid composition, molecular weight, molar absorption coefficients
for denatured (4) and native (5) states, and isoelectric point (6) (see Note 5).
Furthermore, the sequence can be searched for the occurrence of sequence motifs.