How is DNA sequencing performed using Sanger’s dideoxy technique? Give one limitation of this technique.
How is DNA sequencing performed using Sanger’s dideoxy technique? Give one limitation of this technique.
Answer
DNA Sequencing Using Sanger’s Dideoxy Technique:
1. Preparation: DNA is first denatured into single strands.
2. Extension: The single-stranded DNA is then mixed with a DNA polymerase, a primer, and a mixture of normal deoxynucleotides and chain-terminating dideoxynucleotides (ddNTPs).
3. Chain Termination: The ddNTPs cause termination of DNA synthesis at various points.
4. Separation: The resulting fragments are separated by size using capillary electrophoresis.
5. Detection: The fragments are then detected and sequenced based on their fluorescence.
Limitation: Limited by the length of DNA it can sequence (typically up to ~1000 base pairs) and is less efficient for sequencing long genomes.
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