Sanger Sequencing Genetic Analysis

Sanger sequencing remains a fundamental tool in molecular biology, prized for its exceptional accuracy and specificity across various applications. Thermo Fisher Scientific’s Applied Biosystems™ platforms, including the SeqStudio™ Genetic Analyzer, provide integrated sequencing and fragment analysis solutions. Below is an overview of its major applications: 

Core Characteristics of SeqStudio Genetic Analyzer:

• Accuracy >99.99% for single reads.
• Still considered the gold standard for sequencing short DNA regions.
• Performs both Sanger sequencing and fragment analysis without reconfiguration.
• Supports applications like de novo sequencing, targeted resequencing, plasmid sequencing, CRISPR-Cas9 editing confirmation, cell line authentication, and SNP detection.
• Completes runs in as little as 30 minutes, enhancing lab efficiency. 

Key Applications of SeqStudio Genetic Analyzer

1. DNA Sequencing

Sanger sequencing is widely used for sequencing DNA fragments with high precision. It is particularly useful for small-scale sequencing projects where accuracy is essential. It plays a key role in validating mutations or variants detected through other sequencing methods like next-generation sequencing (NGS). Additionally, it complements high-throughput technologies in reference genome sequencing.

2. Targeted Resequencing

Sanger sequencing is crucial for confirming specific genetic variations. It ensures accurate characterization of identified mutations in disease-related genes. It is also used to analyze specific gene regions, such as highly variable genes like HLA or mitochondrial DNA. In clinical genetics applications, targeted resequencing helps pinpoint disease-causing mutations efficiently.

3. DNA Barcoding

Sanger sequencing is a standard method for DNA barcoding, aiding in species identification and biodiversity studies. It is commonly used to identify unknown organisms using genetic markers like COI for animals or rbcL/matK for plants. DNA barcoding facilitates taxonomic classification, allowing researchers to differentiate between closely related species. Additionally, it plays an important role in environmental and conservation studies by helping to monitor species diversity and track invasive organisms.

4. Fragment Analysis

Fragment analysis involves separating and detecting fluorescently labelled DNA fragments, typically from PCR products, to determine their size and quantity. This method is essential for genotyping, microsatellite analysis, and mutation detection. SeqStudio Genetic Analyzer streamlines the process, enabling sequencing and fragment analysis to occur simultaneously without requiring system reconfiguration, thanks to its universal polymer cartridge.

5. Multiplex Ligation-dependent Probe Amplification (MLPA)

MLPA is used to identify copy number variations (CNVs) in genomic DNA. Thermo Fisher’s platforms support MLPA by facilitating the separation of MLPA products via capillary electrophoresis. The resulting data can be analyzed using Coffalyser.Net software to detect genomic deletions or duplications with precision.

6. Minor Variant Detection

Detecting low-frequency variants is critical in areas such as oncology and infectious disease research. Thermo Fisher’s Minor Variant Finder Software improves the sensitivity of Sanger sequencing, enabling the identification of somatic variants present at frequencies as low as 5%. This provides a cost-effective means for confirming variants detected through next-generation sequencing (NGS) or for targeted mutation analysis.

7. Cell Line Authentication

Ensuring accurate cell line authentication is vital for maintaining experimental validity. The SeqStudio Genetic Analyzer, coupled with GeneMapper Software, facilitates efficient STR profiling, aiding in the detection of misidentification or cross-contamination. 

Conclusion: The SeqStudio platform streamlines Sanger sequencing by integrating sequencing and fragment analysis into a single system. Its accuracy, automation, and ease of use make it a valuable tool for genetic research, ensuring efficiency in applications like mutation validation, targeted resequencing, and DNA barcoding. Despite advances in next-generation sequencing, SeqStudio continues to offer reliability and precision in molecular biology and clinical diagnostics.