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Nucleotide Exchange and Excision Technology Homepage

 

Nucleotide Exchange and Excision Technology (NExT) is a predictable and reproducible method to fragment DNA. Thus, NExT DNA shuffling is ideally suited for directed evolution experiments and we successfully employed NExT DNA shuffling for the optimization of enzymes. The method is described in two publications (Nucleic Acids Research 2005, Vol. 33, No. 13 e117 and Molecular Methods in Molecular Biology, in press). In short, a gene is amplified using uridinge (dUTP) as an exchange nucleotide for thymidine (dTTP), together with the three other nucleotides. The uracil moiety is then excised with an enzyme, Uracil-DNA-Glycosylase (UDG), and subsequently the phosphate backbone is opened using alkaline hydrolysis (Piperidine or NaOH). The specific ratio of dUTP to dTTP defines the incorporation rate of uracil and thus the resulting statistical size distribution of fragments. To compute fragment size distributions for a given gene depending on the dUTP:dTTP ratio we wrote the program NExTProg.

 

Installation

The program runs under Windows 95, 98, Me, 2000, XP, 2003, x64. To download right-click the link, chose the save as option, and remember the location where it was saved. The program does not require any installation and can be started by a double click. To remove the program just delete the file. The help file can be called from the program only if it is in the same directory. For easy access you can right-click the program and take advantage of the create shortcut option.

 

Program Basics

NExTProg reads DNA sequences via file,load in the pull down menu and allows the user to enter the fraction of dUTP in % of the combined dUTP and dTTP amount/concentration (e.g. 40 ÁM dUTP and 160 ÁM dTTP in the amplification reaction yields a value of 20%). The parameter window is opened by clicking Params. This window may stay open all the time and the values can be changed as desired. Pressing Start initializes a new calculation with the set values. As a result a a bar chart displays the distribution of the relative amount of fragments versus fragment length. Using the view pull down one can toggle between 'mass' distribution corresponding to the fraction of bases present at this length and 'fragment' distribution corresponding to the number of fragments with the given length. The mass distribution is default as it correlates with intensities seen on DNA gels. The program is for double stranded DNA and automatically takes into account the second strand.

 

Advanced Features

Sequence file format: Any plain text format (ASCII) is suitable. The program reads only the letters A, C, G, and T and is not case sensitive. Consequently, numbers and spaces are ignored but text comments should not be present.

Incorporation Factor: We determined the incorporation preference of dUTP over dTTP for the standard Taq polymerase to be about 0.26 (for details please see the publications). If another polymerase is used this value might vary and should be adjusted.

Multiple dUTP fractions: Several dUTP fractions can be computed in one run resulting in overlaid graphics in the order corresponding to the fractions listed. Note that the 'calculate sequences' option is not available, when multiple fractions are calculated

Range start / end: The output of the program can be restricted to a certain fragment size range corresponding to a gel purification step (e.g. a fragment length from 50 to 150 bases). In this case the program also calculates the theoretical loss in %mass, i.e. the amount outside the desired range.

Calculate sequence: When this box is checked the program calculates every possible fragment sequence and the respective likelihood of occurrence, whihc are then displayed in the 'Fragemtns' panel. Identical sequences are combined regardless of the originating DNA strand. Note that this option becomes increasingly CPU demanding with sequence length and generates very large outputs. If a size range is set only the sequences and relative probabilities within this size range are calculated.

Output panel, scrolling, and data export: Besides the graphical output the programs lists in the lower panels all individual data. To see text exceeding the size of the window, click in the text and use the cursor to scroll left and right or up and down. Within the panel data can be marked and copied. In addition, the File ExportData option allows for the export of data. Exported data can be e.g. imported in spread sheet programs to generate advanced graphics and printouts.

Windows resizing and zoom: All panels can be resized to user needs by grabbing the borders. Pulling a rectangle in the graphic output zooms in the respective area, a right-click in the graphic enables zoom reset. Depending on the settings the very right or left bar of the graphic output might be displayed under the axis or border. In this case zooming in the area visualizes these values.

Logarithmic view: The fragment size axes in the graphical output can be set to a logarithmic scale in order to mimic the running behavior of DNA during electrophoresis.

Please also refer to the help file in rich text format.


Download:

NExTProg 1.0 for Windows: NExTProg.exe

NExTProg info file: NExTinfo.rtf

Sequence example file: cat1_wt.txt

 

Screenshots

Main window with default settings and the test sequence:

NExTProg user interface

 

Parameter window:

NExTProg parameter window

 

Main window with fragment size range set from 50 to 100 bases and calculated sequences:

(Note: There are several sequence distinct fragments with length 50 in the 'Fragments' panel, which are listed combined as length 50 in the 'Distribution' panel. The probability in the 'Fragments' panel depends on the size selection, whereas %mol and %mass are given for the entire sequence. Only a small part of fragment sequences is visible, to see the others one can scroll in the panel or use the export option.) 

 

To get used to the program you can ask for easy to follow tasks, such as range 1 to 1 or range 657 to 657, which is the length of the test sequence. To verify the output you can also load simple sequences such as GTGG or TTTT. Have fun!

 

For questions, problems, or further information please contact Kristian Müller (kristian@biologie.uni-freiburg.de).