Exploring the Classic Sanger Sequencing Method

The video tutorial explains DNA sequencing, focusing on the Sanger method developed in 1977. It covers the basic requirements for in vitro DNA synthesis, emphasizing the role of primers and DNA polymerase. The tutorial details the use of dideoxynucleotides in the Sanger method, which leads to chain termination, allowing for the determination of DNA sequences. The procedure involves four parallel reactions, each with a different dideoxynucleotide, and the results are interpreted using gel electrophoresis. The video concludes with a demonstration of reading and interpreting the autoradiograph to determine the DNA sequence.

What is the primary purpose of DNA sequencing?

• To identify the sequence of nucleotides in a DNA segment

• To determine the order of amino acids in a protein

• To synthesize RNA from a DNA template

• To replicate DNA in a living organism

Who developed the Sanger method of DNA sequencing?

• Rosalind Franklin

• Frederick Sanger

• Francis Crick

• James Watson

What is the role of a primer in DNA synthesis?

• To provide a starting point for new strand synthesis

• To break down the DNA strand

• To terminate DNA synthesis

• To act as a template for DNA polymerase

Why is a primer essential for DNA polymerase to function?

• It provides the 5' end for DNA polymerase

• It stabilizes the DNA strand

• It acts as a catalyst for the reaction

• It provides the 3' hydroxyl group for phosphodiester bond formation

What is a dideoxynucleotide (ddNTP)?

• A nucleotide with an extra hydroxyl group

• A nucleotide that lacks a 3' hydroxyl group

• A nucleotide that forms a double bond with the template strand

• A nucleotide that enhances DNA synthesis

What happens when a ddNTP is incorporated into a growing DNA strand?

• DNA strand forms a loop

• DNA synthesis terminates

• DNA synthesis continues normally

• DNA synthesis is enhanced

How many separate reactions are set up in the Sanger sequencing method?

• Two

• Three

• Four

• Five

What is the purpose of adding small amounts of ddNTPs in each reaction?

• To enhance DNA synthesis

• To occasionally terminate DNA synthesis

• To act as a template for DNA polymerase

• To stabilize the DNA strand

What technique is used to separate the partial DNA fragments in Sanger sequencing?

• Chromatography

• Centrifugation

• Polyacrylamide gel electrophoresis

• Agarose gel electrophoresis

How is the sequence of the DNA segment determined in Sanger sequencing?

• By measuring the intensity of the bands

• By reading the sequence from bottom to top of the autoradiograph

• By counting the number of bands in the gel

• By reading the sequence from top to bottom of the autoradiograph