In a dideoxy DNA sequencing experiment, four separate reactions are carried out to provide the replicated material for DNA sequencing gels. Reaction products are usually run in gel lanes labeled A, T, C, and G.
Identify the nucleotides used in the dideoxy DNA sequencing reaction that produces molecules for the A lane of the sequencing gel.

Verified step by step guidance

1. Understand the principle of dideoxy DNA sequencing (Sanger sequencing): This method uses a DNA template, a primer, DNA polymerase, deoxynucleotides (dNTPs), and dideoxynucleotides (ddNTPs). The ddNTPs lack a 3'-OH group, which terminates DNA synthesis when incorporated.
2. Recognize that the A lane of the sequencing gel corresponds to DNA fragments that terminate at adenine (A) in the template strand. This means the complementary nucleotide (thymine, T) is incorporated in the newly synthesized strand.
3. Identify the nucleotides required for the A lane reaction: The reaction mixture must include all four deoxynucleotides (dATP, dTTP, dCTP, dGTP) to allow normal DNA synthesis, as well as a small amount of dideoxyadenosine triphosphate (ddATP) to terminate synthesis at positions complementary to adenine in the template strand.
4. Understand the role of ddATP: When ddATP is incorporated into the growing DNA strand, it prevents further elongation because it lacks the 3'-OH group necessary for forming a phosphodiester bond with the next nucleotide.
5. Conclude that the A lane reaction contains the following nucleotides: dATP, dTTP, dCTP, dGTP, and ddATP. The ddATP ensures that DNA fragments of varying lengths are generated, each terminating at a position complementary to adenine in the template strand.

Key Concepts

Dideoxy Nucleotide Triphosphates (ddNTPs)

Dideoxy nucleotide triphosphates (ddNTPs) are modified nucleotides used in DNA sequencing. Unlike regular deoxynucleotides, ddNTPs lack a hydroxyl group at the 3' position, preventing further elongation of the DNA strand once incorporated. This property allows for the termination of DNA synthesis at specific bases, which is crucial for determining the sequence of nucleotides in the DNA template.

Chain Termination Method

The chain termination method is a technique used in dideoxy DNA sequencing where DNA polymerase synthesizes new DNA strands. In this method, a mixture of regular deoxynucleotides and ddNTPs is used, leading to the production of DNA fragments of varying lengths that terminate at specific nucleotides. These fragments are then separated by size through gel electrophoresis, allowing for the identification of the sequence based on the terminated bases.

Gel Electrophoresis

Gel electrophoresis is a laboratory technique used to separate DNA fragments based on their size. In the context of dideoxy sequencing, the terminated DNA fragments are loaded into a gel matrix and subjected to an electric field, causing smaller fragments to migrate faster than larger ones. The resulting pattern of bands corresponds to the different lengths of DNA fragments, which can be visualized and analyzed to determine the nucleotide sequence of the original DNA template.