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How are nucleotides with dideoxyribonucleic acid used to stop DNA replication for base sequencing?
How are nucleotides with dideoxyribonucleic acid used to stop DNA replication for base sequencing?
Answered
Dideoxyribonucleic acid (ddNTPs) are crucial components in the process of DNA sequencing, particularly in the Sanger method, which utilizes chain-terminating nucleotides to stop DNA replication at specific points. Here’s how ddNTPs function in this context:
Mechanism of ddNTPs in DNA Sequencing
- Structure of Dideoxynucleotides:
- Dideoxynucleotides lack a 3′-hydroxyl group (-OH) on the sugar component of the nucleotide. This structural difference is critical because it prevents the formation of a phosphodiester bond with the next incoming nucleotide during DNA synthesis.
- Chain Termination:
- When a ddNTP is incorporated into a growing DNA strand by DNA polymerase, it terminates further elongation of that strand. Since there is no 3′-OH group available for the next nucleotide to attach, the addition of any further nucleotides is halted. This characteristic allows researchers to generate fragments of varying lengths that terminate at specific nucleotides.
- Sanger Sequencing Process:
- In Sanger sequencing, a single-stranded DNA template is mixed with normal deoxynucleotides (dNTPs) and one type of ddNTP (either ddATP, ddTTP, ddCTP, or ddGTP) in separate reaction mixtures. Each mixture corresponds to one of the four bases.
- As DNA polymerase extends the primer from the template strand, it incorporates both dNTPs and ddNTPs randomly. The incorporation of a ddNTP results in a terminated fragment.
- Fragment Generation:
- The result is a collection of DNA fragments that vary in length, each ending with a specific ddNTP that indicates the corresponding base in the original template strand. For example, if a ddGTP is incorporated, it signifies that the last base added was cytosine (C), as G pairs with C.
- Separation and Analysis:
- After the reaction, these fragments are separated by size using gel electrophoresis or capillary electrophoresis. The shortest fragments migrate the farthest through the gel.
- By analyzing the pattern of terminated fragments, researchers can deduce the sequence of bases in the original DNA template.
- Detection Methods:
- To facilitate detection, ddNTPs can be labeled with fluorescent dyes or radioactive markers. Automated sequencers can then read these labels to determine the sequence based on fragment length and color.
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