Cycle sequencing produces linear amplification of extension products, specifically DNA fragments of varying lengths that are labeled with fluorescent tags. These fragments are essential for determining the nucleotide sequence of a DNA template.
Here's a breakdown of the process and the products:
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The Process: Cycle sequencing is a thermal cycling technique similar to PCR, but it uses only one primer. This leads to linear, rather than exponential, amplification. It involves repeated cycles of:
- Denaturation: Heating the DNA template to separate the strands.
- Annealing: Cooling the mixture to allow the primer to bind to the template.
- Extension: DNA polymerase extends the primer, adding nucleotides to create a new DNA strand.
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Key Ingredients:
- DNA Template: The DNA region you want to sequence.
- Primer: A short DNA sequence that initiates DNA synthesis. Only one primer is used per reaction, leading to linear amplification.
- DNA Polymerase: An enzyme that adds nucleotides to the growing DNA strand.
- Deoxynucleotide triphosphates (dNTPs): Normal DNA building blocks (dATP, dGTP, dCTP, dTTP).
- Dideoxynucleotide triphosphates (ddNTPs): Modified nucleotides that, when incorporated into the growing DNA strand, terminate extension. Crucially, these are fluorescently labeled, each ddNTP base (A, T, C, G) having a different fluorescent dye.
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The Products:
- DNA Fragments of Varying Lengths: The key output. Because ddNTPs randomly terminate extension, the reaction produces a population of DNA fragments, each ending with a fluorescently labeled ddNTP. The fragments differ in length by one nucleotide.
- Fluorescently Labeled Fragments: Each fragment ends with a ddNTP that's tagged with a specific fluorescent dye, allowing for detection.
- Linear Amplification: Unlike PCR, the amplification is linear, meaning each cycle produces a relatively constant number of new DNA strands complementary to the template. This is because only one primer is used.
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Purpose: The resulting collection of fluorescently labeled DNA fragments are then separated by size using capillary electrophoresis. As each fragment passes a detector, the fluorescent dye is read, allowing the base at the end of the fragment to be identified. By ordering the fragments by size, the complete DNA sequence can be determined.
In summary, cycle sequencing generates a collection of fluorescently labeled DNA fragments of varying lengths, representing all possible termination points in the DNA sequence, which are then used to determine the overall sequence.
