Fragment Analysis -- the Other Half of your Applied Biosystems' Genetic Analyzer

Summary

This video highlights how genetic analyzers, typically used for Sanger sequencing, can also be employed for fragment analysis in diverse applications. It explains the process of analyzing DNA fragments using PCR, fluorescent labeling, and capillary electrophoresis, and emphasizes the system's versatility in multiplexing, sensitivity, and ease of use. The video covers real-world applications such as microsatellite analysis for paternity testing, MLPA for gene copy number variations, and SNP analysis using the Snapshot kit. It also introduces custom fragment analysis techniques for various laboratory needs, showcasing the power and flexibility of the genetic analyzer.

Takeaways

• 😀 Genetic analyzers, often known for Sanger sequencing, can also be used for fragment analysis to determine DNA fragment sizes.
• 😀 PCR-generated DNA fragments are mixed with a size standard and injected into the genetic analyzer for size-based separation.
• 😀 Smaller DNA fragments migrate faster than larger ones through the capillary during electrophoresis.
• 😀 Fluorescent signals from DNA fragments are detected, generating peaks in an electropherogram, which are analyzed by GeneMapper software.
• 😀 A sizing curve is created using known DNA fragment sizes to accurately determine the size of unknown fragments.
• 😀 Multiplexing is possible with genetic analyzers, allowing for analysis of multiple DNA fragments in a single sample using different fluorescent dyes.
• 😀 The analyzer can handle overlapping fragment size ranges, with fluorescent multiplexing allowing simultaneous detection of multiple dyes.
• 😀 Genetic analyzers are highly sensitive and can work with difficult DNA samples, such as blood spots or formalin-fixed tissues.
• 😀 Fragment analysis requires minimal sample preparation compared to sequencing, making it a more straightforward approach.
• 😀 Common fragment analysis applications include microsatellite analysis (used in paternity testing and forensics), gene copy number variation (MLPA), and SNP analysis.
• 😀 Fragment analysis can also be used to compare entire genomes (e.g., AFLP method), providing insights into genetic variation without needing the full DNA sequence.

Q & A

What is the primary use of genetic analyzers in molecular biology?

Genetic analyzers are primarily used for Sanger sequencing, a method for sequencing DNA fragments. However, they can also be used for fragment analysis, which involves determining the size of DNA fragments.

How is the size of an unknown DNA fragment determined in fragment analysis?

The size of an unknown DNA fragment is determined by comparing its migration time in a capillary electrophoresis system to a sizing curve created using DNA fragments of known sizes.

What is the role of fluorescent labels in fragment analysis?

Fluorescent labels are used to tag both the unknown DNA fragments and the size standards. The system detects the fluorescence to generate peaks in an electropherogram, which are then analyzed to determine the size of the unknown fragments.

What is multiplexing in the context of fragment analysis?

Multiplexing allows the simultaneous analysis of multiple DNA fragments of similar or overlapping size ranges by using different fluorescent dyes. This enables the detection of multiple fragments in a single run.

What are some of the advantages of using a genetic analyzer for fragment analysis?

Genetic analyzers offer high sensitivity, the ability to work with various DNA templates (even difficult ones), and straightforward data interpretation, making them ideal for diverse fragment analysis applications.

How does fragment analysis benefit applications like paternity testing?

In paternity testing, fragment analysis is used to compare micro satellite profiles between parents and children. The inherited micro satellite alleles in the child are identified by determining the size of the DNA fragments containing the repeats.

What is MLPA, and how does it relate to fragment analysis?

MLPA (Multiplex Ligation-dependent Probe Amplification) is used in fragment analysis to determine gene copy number variations in cancer samples. It works by hybridizing probes to target genes and measuring fragment intensity to assess gene copy numbers.

How can fragment analysis be used to study single nucleotide polymorphisms (SNPs)?

Fragment analysis of SNPs involves using a primer to hybridize to the region before the SNP site, performing a primer extension reaction with fluorescently labeled ddNTPs, and analyzing the labeled fragments to identify the SNP variation.

What is the AFLP method, and how does it use fragment analysis?

The AFLP (Amplified Fragment Length Polymorphism) method is used to compare genomes without prior knowledge of the sequences. It involves cutting the genome with restriction enzymes, amplifying the fragments, and comparing the peak profiles to identify genetic variation.

Can custom fragment analysis applications be developed using genetic analyzers?

Yes, genetic analyzers allow researchers to develop custom fragment analysis applications by selecting appropriate fragment sizes, fluorescent labels, and other parameters suited to specific research needs.