Sanger sequencing

Definition

Sanger sequencing is a method used to determine the precise order of nucleotides in a DNA molecule by incorporating chain-terminating dideoxynucleotides during DNA replication. This technique, developed by Frederick Sanger in the 1970s, allows for the accurate sequencing of both short and long stretches of DNA, making it a foundational tool in genetics and molecular biology for analyzing DNA structure and sequencing.

5 Must Know Facts For Your Next Test

1. Sanger sequencing uses a combination of normal deoxynucleotides and dideoxynucleotides to create fragments of varying lengths, each ending with a labeled dideoxynucleotide that represents the last base in the sequence.
2. The method involves running these DNA fragments through gel electrophoresis, allowing researchers to visualize the length of each fragment and thus deduce the DNA sequence based on the labeled nucleotides.
3. Sanger sequencing is known for its high accuracy and was the method used in the Human Genome Project for sequencing human DNA.
4. While Sanger sequencing has largely been replaced by next-generation sequencing technologies for large-scale projects, it remains widely used for smaller-scale applications and validation of next-generation sequencing results.
5. The original Sanger sequencing method can read sequences up to about 800-1000 base pairs long, which is suitable for many practical applications in molecular biology.

Review Questions

• How does Sanger sequencing utilize dideoxynucleotides to determine the sequence of DNA?
  • Sanger sequencing incorporates dideoxynucleotides during the DNA synthesis process. These special nucleotides lack a 3' hydroxyl group, which means that once they are added to a growing DNA strand, no further nucleotides can be attached. This results in fragmented DNA strands that terminate at each incorporation of a dideoxynucleotide, effectively allowing researchers to identify the sequence based on the lengths of these fragments and their associated labels.
• Discuss the role of gel electrophoresis in the Sanger sequencing process and how it contributes to interpreting results.
  • Gel electrophoresis plays a critical role in Sanger sequencing by separating the various DNA fragments generated during the reaction. Once the fragments are created, they are loaded onto a gel matrix where an electric current is applied. The smaller fragments move faster than larger ones, allowing researchers to separate them based on size. This separation enables the visualization of bands corresponding to different fragment lengths, making it possible to read the DNA sequence from the shortest to longest fragment.
• Evaluate how Sanger sequencing has influenced modern genomic research compared to newer technologies.
  • Sanger sequencing laid the groundwork for modern genomic research by providing a reliable and accurate method for determining DNA sequences. Its development was crucial for projects like the Human Genome Project, where high-quality sequence data was paramount. Although next-generation sequencing technologies have significantly increased throughput and reduced costs, Sanger sequencing still holds importance for its precision in validating sequences obtained through these newer methods. The combination of both approaches allows researchers to ensure accuracy while exploring large genomic datasets.

Related terms

Dideoxynucleotide: A type of nucleotide used in Sanger sequencing that lacks a 3' hydroxyl group, causing DNA synthesis to terminate when incorporated into a growing DNA strand.

Polymerase Chain Reaction (PCR): A method used to amplify specific segments of DNA, making it easier to analyze and sequence them using techniques like Sanger sequencing.

Gel electrophoresis: A laboratory technique used to separate DNA fragments based on their size, which is essential for visualizing the results of Sanger sequencing.