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Rapid, High Throughput Mutation Discovery Using Fluorescent SSCP on the ABI PRISM 3100 and 3100Avant Genetic Analyzers

Abstract

Single-strand conformation polymorphism (SSCP) analysis on the ABI PRISM 3100 and the 3100 Avant Genetic Analyzers provides fast, easy detection of small variations in DNA sequences, including singlebase substitutions, insertions, or deletions.

SSCP Analysis

SSCP analysis is based on the principle that under non-denaturing conditions, single-stranded DNA molecules assume unique conformations that vary depending on their nucleotide sequence. The change of only one base may cause a conformational change in the DNA molecule that is sufficient to be detected on a non-denaturing polymer, e.g., electrophoretic mobility differences compared with wild-type (wt) sequences.

The SSCP technique includes three processes:

PCR amplification using primers that flank the DNA region of interest;

Denaturation of the resulting double-stranded PCR product, followed by rapid chilling to prevent re-annealing of the strands;

Electrophoretic separation of the single-stranded DNA on a nondenaturing sieving medium, such as a flowable polymer.

The ABI PRISM 3100 Genetic Analyzer, a 16-capillary instrument, performs fluorescent SSCP rapidly and efficiently, separating fragments of up to 350 bases in less than 35 minutes with a minimum throughput of 654 samples per 24 hours. Fluorescent SSCP analysis on the 3100 system can be an ideal first-pass discrimination test for screening a large number of samples. Subsequent sequence analysis on this instrument can determine the exact base change of the mutation. This can be much more cost-effective than sequencing every sample, especially for populations in which the mutation frequency is low.

For studies involving fewer samples and lower throughput, the ABI PRISM 3100 Avant Genetic Analyzer is suited. A four-capillary instrument, the 3100 Avant instrument includes the same design features as the 3100 instrument, providing comparable performance at lower throughput. By utilizing this four-capillary instrument for mutation detection, users can analyze up to 163 samples in a 24-hour period.

Benefits of SSCP Analysis on the ABI PRISM 3100 and 3100 Avant Systems

In addition to optimal throughput for all applications, the ABI PRISM 3100 and 3100 Avant Genetic Analyzers offer the following benefits for SSCP analysis:

Analysis at varying temperatures

The 3100 and 3100 Avant analyzers provide automated electrophoresis of a single sample at stable, uniform temperatures ranging from 18C to 65C. Running samples at varying temperatures maximizes the probability of detecting novel mutations. This flexibility allows researchers to determine the optimal temperature for differentiating mutant and wild-type strands.

Fluorescent labeling of forward and reverse strands

The ABI PRISM 3100 and 3100 Avant multi-fluorescence systems offer unique differential labeling of forward and reverse strands. Four-color fluorescent labeling eliminates the safety concerns inherent in isotopic-dye systems while increasing the probability of detecting novel mutations and reducing error. For example, under a given condition, more than one stable conformation of each DNA strand may exist. Labeling the two strands with different colors simplifies data comparison and ensures that the same strands are being compared from sample-to-sample. In addition, residual double-stranded products display both colors and are easily distinguished from single-stranded DNA molecules. Overlapping single strands may also show this pattern.

Internal size standards for detecting mobility differences

The availability of multiple dyes permits the use of internal size standards for normalization of mobility rates. The internal size standards align data in various capillaries, thus eliminating capillary- to-capillary or run-to-run variability. This ensures the accuracy and precision that are critical for successful SSCP analysis.

Analysis at varying temperatures

The 3100 and 3100 Avant analyzers provide automated electrophoresis of a single sample at stable, uniform temperatures ranging from 18C to 65C. Running samples at varying temperatures maximizes the probability of detecting novel mutations. This flexibility allows researchers to determine the optimal temperature for differentiating mutant and wild-type strands.

Fluorescent labeling of forward and reverse strands

The ABI PRISM 3100 and 3100 Avant multi-fluorescence systems offer unique differential labeling of forward and reverse strands. Four-color fluorescent labeling eliminates the safety concerns inherent in isotopic-dye systems while increasing the probability of detecting novel mutations and reducing error. For example, under a given condition, more than one stable conformation of each DNA strand may exist. Labeling the two strands with different colors simplifies data comparison and ensures that the same strands are being compared from sample-to-sample. In addition, residual double-stranded products display both colors and are easily distinguished from single-stranded DNA molecules. Overlapping single strands may also show this pattern.

Internal size standards for detecting mobility differences

The availability of multiple dyes permits the use of internal size standards for normalization of mobility rates. The internal size standards align data in various capillaries, thus eliminating capillary- to-capillary or run-to-run variability. T his ensures the accuracy and precision that are critical for successful SSCP analysis.

Labeling methods Dye-labeled primers increase sensitivity.

Dye sets DS-30 (6FAM, HEX, NED, ROX dye) and DS-31 (6FAM, VIC, NED, ROX dye) offer good spectral resolution and are recommended. Applied Biosystems provides custom nucleic acid synthesis services that will synthesize the primers for you. To find out more, please visit our website at www.appliedbiosystems.com.

ABI PRISM 3100 and 3100 Avant Genetic Analyzers

Key Features Versatility

Variable temperature control from 18C to 65C to optimize analysis

Multiple dye selection for optimal throughput and sensitivity

Automated sample injection from two 96-well microtiter plates for the 3100 system and one 96-well microtiter plate for the 3100 Avant system

Selection of capillary lengths, separation matrices including GeneScan software native polymers for SSCP and chemistry kits

Support for full range of fragmentanalysis and sequencing applications

SSCP Throughput ABI PRISM 3100 Genetic Analyzer

16 capillaries for high throughput applications

654 samples in 24 hours

ABI PRISM 3100 Avant Genetic Analyzer

4 capillaries for low-to-medium throughput applications

163 samples in 24 hours

Ease-of-use

Fully automated polymer filling, sample injection, detection, and data analysis

24-hour unattended operation

Easy instrument set-up

Convenience

Minimal hands-on labor; no slab gels to prepare, no loading with pipettes, no glass plates to clean, no sample lanes to track

Windows platform for analysis and results editing

Easy, direct transfer of files for further analysis and reporting

Data collection and analysis software

Applied Biosystems provides datacollection and analysis software for sequencing and genotyping applications. SSCP analysis on both platforms requires GeneScan or GeneMapper software.

GeneScan Analysis and GeneMapper Software

Key Features

Provides powerful tool for mutation discovery

Analyzes collected data, automatically identifies peaks, measures peak intensity, and sizes each DNA fragment

Corrects for run-to-run variability to ensure accurate and reproducible data and simplified calculation of mobility shifts detected with SSCP

Allows analysis of individual files or batch-processing of several runs; displays or prints data in multiple configurations

Conclusion

Fluorescent SSCP, now available on the ABI PRISM 3100 and the 3100 Avant Genetic Analyzers, is a fast, versatile, and accurate method of screening large numbers of samples for novel mutations. It is an excellent method of saving researchers time and money normally spent sequencing initial samples.

References

Atha DH, Wenz HM, Morehead H, Tian J, and OConnell CD. Detection of p53 point mutations by single-strand conformation polymorphism: analysis by capillary electrophoresis. Electrophoresis . 1998 Feb. 19(2):172179.

Hayashi, K., et al . PCR-SSCP: A method for detection of mutations. 1992 GATA 9:7379.

Wenz HM, Baumhueter S, Ramachandra S, and Worwood M. A rapid automated SSCP mu ltiplex capillary electrophoresis protocol that detects the two common mutations implicated in hereditary hemochromatosis (HH). Hum Genet . 1999 Jan. 104(1):2935.

Ancillary Items

100% w/w glycerol

10xTBE

0.3 N sodium hydroxide, stored in a plastic container

Deionized water (microbiology grade)


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