High Throughput Mutation Detection Using Capillary Electrophoresis-Based

Single Strand Conformational Polymorphism (CE-SSCP) Analysis

Overview

Under non-denaturing conditions, single-stranded DNA (ssDNA) molecules uniquely conform depending on their nucleotide sequence.  A single base change may cause a difference in the conformation that can be detected on a non-denaturing polymer. This is the basis for single-strand conformational analysis, a method to discover mutations and  polymorphisms in DNA.

Using PCR primers that flank the region of interest, both the forward and reverse strands are fluorescently labeled.  Following amplification, the PCR products are denatured to separate the strands, then rapidly chilled to keep them from re-annealing.  The ssDNA is then electrophoresed on a non-denaturing polymer that shows the individual mobility of each labeled strand.

Figure 1:  Diagram illustrating the process and theory behind CE-SSCP

The figure above illustrates the CE-SSCP process.  Once labeled with fluorescent forward and reverse primers, the strands are mixed with a size standard (not shown). The size standard consists of PCR products of known sizes that are used during data analysis to uniformly align the sample data.

The samples are then electrokinetically injected into a capillary containing a non-denaturing polymer.  An electric current is applied, and the fluorescent single DNA strands are electrophoresed through the polymer and detected by laser-excitation coupled with a CCD camera. An Applied Biosystems (ABI) model 3100 DNA Analyzer performs the electrophoresis. Changes in single strand conformation will be revealed by changes in mobility of the separated DNA strands.

The ABI Prism 3100 Genetic Analyzer has the capacity to perform multi-temperature runs from 18 - 65 degrees Celsius.  Since a mutation may display a mobility shift at one temperature and not another, the samples are run at multiple temperatures to screen for mutations with greater sensitivity (~ 98%).

CE-SSCP Data for Exon 18

 of the CLCN2 gene

The figure below demonstrates data from a routine CE-SSCP run.  The wild type DNA sample is illustrated in the upper panel and the mutant DNA sample in the lower panel.  The arrows in the lower panel indicate the extra peaks shown by the mutant.

Figure 2:  CE-SSCP data for Exon 18 of the CLCN2 gene showing an abnormality in the lower panel.  This data is analyzed and presented using ABI GeneScan software

Once a change in the peak morphology (such as in the example above) is detected, selective sequencing can be done on samples that exhibit abnormal peak conformations.  These sequences can then be aligned and compared to those of reference wild-type sequences. 

The figure below shows both the wild type and mutant samples from the CE-SSCP data presented above.

Figure 3:  A segment of the sequence from Exon 18 of the CLCN2 gene revealing a single base change that confirms the CE-SSCP results

In the lower panel, the sequence reveals that the individual is heterozygous for a C/T base change.