Single-Stranded Conformation Polymorphism (SSCP)

Figure 1: Mutation Screening

Many researchers in the field of genomics need to be able to reliably identify allelic variants of genetic traits. Single-stranded conformation polymorphism (SSCP) is a widely used technique that can be an effective screening tool to find variants in a large number of samples, in everything from micro-organisms to humans. The SSCP analysis method detects point mutations by analyzing their unique electrophoretic mobility that results from small changes in the variant's nucleotide sequences.

Mutation screening using SSCP analysis on an Applied Biosystems Genetic Analyzer and GeneMapper® Software (Figure 1). Gray bands indicate the expected peaks for three known mutations.

Products
Experimental Workflow: Find the Products You Need for Every Step
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Step 1: DNA Purification

DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction. Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction.

Products for Step 1: DNA Purification

NucPrep® DNA Chemistry for Tissues

Starter kit for DNA isolation from animal and plant tissues and cells, etc., using NucPrep® Chemistry.

MagMAX™ Express Magnetic Particle Processor

MagMAX™ Express magnetic particle processors are ideal for researchers who want to automate their MagMAX sample preparation kits. The MagMAX Express platform is the premier automated platform to meet your throughput needs.

MagMAX™ Express-96 Magnetic Particle Processors

MagMAX™ Express magnetic particle processors are ideal for researchers who want to automate their MagMAX sample preparation kits. The MagMAX Express platform is the premier automated platform to meet your throughput needs.

Step 2: Primer Design & PCR Amplification

PCR amplify loci of interest with fluorescently labeled primers.

Products for Step 2: Primer Design & PCR Amplification

GeneAmp® Gold RNA PCR Reagent Kit and Core Kit

The GeneAmp® Gold RNA PCR Reagent Kit has been optimized to combine reverse transcription using MultiScribe™ Reverse Transcriptase, with highly sensitive and specific amplification using AmpliTaq Gold® DNA Polymerase.

Thermocycler Systems

Step 3: Sample Prep for Fragment Analysis Run

Add aliquot of PCR reaction to size standard and Hi-Di Formamide mix; heat denature, chill to 4°C and proceed with electrophoresis.

Products for Step 3: Sample Prep for Fragment Analysis Run

Hi-Di™ Formamide

Highly deionized formamide used to resuspend samples before electrokinetic injection in capillary electrophoresis systems.

GeneScan™ Size Standards

GeneScan™ Size Standards are fluorescently-labeled DNA fragments required for performing fragment analysis and genotyping applications on our capillary electrophoresis platforms.

Step 4: Capillary Electrophoresis

During capillary electrophoresis, the products of the PCR reaction are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the negatively charged DNA fragments move through the polymer in the capillaries toward the positive electrode.

Products for Step 4: Capillary Electrophoresis

3500 Genetic Analyzer

The 3500 8-capillary platform can run a wide variety of applications—including de novo sequencing and resequencing (mutational profiling)—as well as microsatellite analysis, MLPA™, LOH, MLST, AFLP® and SNP validation or screening. The majority of applications can be run on a single polymer and capillary array.

3500xl Genetic Analyzer

The 3500xL 24-capillary platform can run a wide variety of applications, including de novo sequencing and resequencing (mutational profiling), as well as microsatellite analysis, MLPA™, LOH, MLST, AFLP® and SNP validation or screening. The majority of applications can be run on a single polymer and capillary array.

3730 DNA Analyzer

The 48-capillary 3730 DNA Analyzer is the Gold Standard in medium-to-high throughput genetic analysis. Use this for DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection and traditional DNA sequencing. Get the highest quality data at a low cost per sample. This is upgradeable to 96 capillaries.

3730xl DNA Analyzer

The 96-capillary 3730xl DNA Analyzer is the Gold Standard for high throughput genetic analysis. Use this for DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection and traditional DNA sequencing. Get the highest quality data at a low cost per sample.

3130 Genetic Analyzer

Applied Biosystems 3130 Genetic Analyzer is the latest generation of 4-capillary electrophoresis instruments for the low to medium throughput laboratories. The system offers industry-leading performance, plus sophisticated automation capabilities allowing you to save time, reduce costs and increase productivity.

3130xl Genetic Analyzer

Applied Biosystems 3130xl Genetic Analyzer is the latest generation of 16-capillary electrophoresis instruments for the medium throughput laboratories.

310 Genetic Analyzer

The ABI PRISM® 310 Genetic Analyzer is an automated single-capillary genetic analyzer designed for a wide range of sequencing and fragment analysis applications.

Step 5: Data Analysis

Use secondary analysis software to detect mutations. SSCP detects mutations in DNA fragments due to electrophoretic mobility shifts caused by changes in the secondary structure of single-stranded DNA fragments. Single-stranded DNA fragments will fold in sequence-specific secondary structures, which usually have individual migration patterns during the capillary electrophoresis. DNA fragments with mutations are detected by a peak pattern that is different compared to the peak patterns in normal control samples.

Products for Step 5: Data Analysis

GeneMapper® Software

GeneMapper is a flexible genotyping software package that provides DNA sizing and quality allele calls for all Applied Biosystems electrophoresis-based genotyping systems.

Peak Scanner™ Software

Use this free software to perform DNA fragment analysis; separate a mixture of DNA fragments according to their sizes, provide a profile of the separation, and precisely calculate the sizes of the fragments.
Literature
Application Note
- Single-Stranded Conformation Polymorphism (SSCP) on Applied Biosystems Capillary Electrophoresis Systems