TGGE DGGE: PCR primer design

Institute for Medical Genetics - Charite Berlin

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Optimization of mutation screening systems with
temperature gradient gel electrophoresis and PCR

Introduction

PCR combined with temperature gradient gel electrophoresis is a rapid and very sensitive screening method for point mutations. It may be used to identify exons or other DNA segments with abnormal electrophoretic mobility which are then subjected to DNA sequencing. Computer aided design of PCR primers for denaturing gradient gel electrophoresis and the careful choice of a suitable temperature range is the most important warranty for the success of the screening. The program was written, to facilitate the design of PCR primers fulfilling the requirements for a sensitive mutation screening. It supports the new concept of bipolar clamping which is important when mono-polar TGGE leads to fuzzy bands.
The MS-DOS program tggedemo.exe starts an animation which explains the melting processes of DNA-fragments during TGGE.

Screen Shots of the analysation of exon 14 of the NF1-gene

The images are quater size only. You get them in FULL SIZE by clicking on them.

Primer-binding sites and self complementarity of primers.

The upper part contains the sequence text. The proposed binding site for the forward primer is marked red and the binding site for the reverse primer green. Below the self complementarities are shown in the 1st and 2nd column and the mutual complementarity is given in the 3rd column. All significant possibilities of primer-primer annealing are listed. The most disturbing cases are on top of the list. They exhibit many hydrogen (yellow ticks). bonds at the 3' ends are most disturbing.

Melting profile

Each tick on the x-axis represents a base pair. The base-coloring is the same as in figure 1a. The base pairs are numbered from 1 to 195. The y-axis shows the temperature where the probability for a base pair to be melted has the value 0.95, 0.75, 0.5, 0.25 and 0.5, respectively. At the 5'-end (left) the thermostability is artificially increased by a clamp and leads to very high temperatures. Apart from the clamp one can distinguish two further melting domains: from the 5'-terminus to the 50th base pair and from the 50th base pair extending to the 3'-terminus. The difference between these two melting domains is little and the sensitivity of TGGE is not disturbed. If the difference between these two plateaus in the curve would be higher, both region had to be tested in two different PCR-TGGE steps. Three asterisks above the x-axis mark positions of mutations detected with this assay. They are included in the sequence text in figure 1a.

Resolving heteroduplices with TGGE

In case of a heterozygous mutation two hetero-duplex bands evolve. They are indicators for mutations. Heteroduplices do not migrate as far as the wild type fragments because they "melt" at lower temperatures. The distance of hetero-duplex bands and wild type bands depends on the electrophoretic duration (x-axis) and the base position (y-axis). A mutation can only be detected, when the displacement is higher than the resolution of the gel.

Bipolar clamping versus monopolar clamping

Bipolar clamping improves bands

Usually only one of both PCR-primers is tagged with a psoralen molecule (monpolar, lanes on the left side). The exon 14 of the fibrillin-gen (Marfan syndrom) could not be analyzed in the usual way because the bands were fuzzy.
We tried to modify both PCR-primers with a psoralen. With bipolar clamping (right) the bands were sharp and the mutations could safely be identified. Besides the wildtype band three additional bands appear: 2 heteroduplex bands, one mutant homoduplex band.

Download and Installation Guide

Windows-XP incompatibility: Since Microsoft's operation systems are widely used, TGGE-Star has been designed for Microsoft operation system. Microsoft does not support source code installation and programs are usually distributed in binary form. This often leads to incompatibility with new versions of the operation system.
TGGE-Star cannot be run directly under Windows-XP any more, but can be started in a DOS emulator such as DosBox.

TGGE-Star comes as a self extracting archive and is installed into the directory c:\set or d:\set depending on the active drive letter. The directory \set\ is demanded for by the included melting analysis module melt87. Nevertheless, by assigning a drive letter to a directory, it is possible to install it anywhere else. No other files on your computer are altered. The registry is not touched. Please run setup.exe. It installes TGGE-STAR in the directory \SET\.

TGGE-STAR may use any text editor or graphic program. These settings are made in the menu preferences.
PLEASE ACKNOWLEDGE ANY RESULTS FROM MELT87 AND SQHTX IF THEY ARE USED IN PUBLICATIONS. These two programs by Lerman and Silverstein are embedded into this package.

MS-DOS	Change into the directory \SET. Start PRIMER.BAT by typing PRIMER at the MS-DOS prompt.
WINDOWS 95/98	Double click on the file C:\SET\PRIMER.BAT. For convenience you can create a symbolic link and place it on your desk. Important Windows9x trick: ALT-RETURN toggles full screen of the MS-DOS-terminal window.
WINDOWS-NT	see WINDOWS 9.x On some machines we had problems. Note: all users of the program need write permission on the directory /set/
WINDOWS-2000	see WINDOWS 9.x Avoid ALT-RETURN. This hangs the terminal window. The DOS-shell is not in the start-up menu. The DOS-shell is a program called CMD.EXE.
Windows-XP	TGGE-STAR is not directly running under Windows-XP any more. Solution: install and run DosBox. This will run TGGE-STAR about 20 times slower, but will be fine on a modern PC.
LINUX	The easiest way to get it running is to install DosBox. The disadvantage of DosBox is that the program will be very slow (5% of the native speed). DOSEMU is more difficult to setup. The advantage is that it will run at full spead. The best is to seek help from your system administrator.

Documentation and hyper links

Other projects:

My web site can be visited here Last modified: Tue Aug 31 19:05:04 CEST 2004