Fluorescent In Situ Hybridization(FISH)
DNA sequencing utilizing fluorescent markers is a method to analyze genetic sequences and visualize specific genes or genomic regions by attaching fluorescent dyes to specific bases. One of the most representative methods includes the following procedure:
- Use of Fluorescent Markers: Each DNA base (A, T, G, C) is tagged with a different fluorescent marker (color). As a result, each base emits light of a specific wavelength, giving it a unique color.
- Sequencing Process: During DNA sequencing, the sequence of a specific DNA fragment is determined. Each DNA base amplified or replicated in the sequencing reaction is identified by the color of light emitted by the fluorescent marker attached to it.
- Image Capture and Analysis: The light emitted by the fluorescent markers is captured using a special camera or detector. This data is analyzed by computer software, determining the DNA sequence based on the colors of the fluorescent markers observed at each position.
- Sequencing Results: Ultimately, the DNA sequence is represented by the order of fluorescent colors. This allows researchers to identify specific areas of the genome, genetic mutations, or genetic changes related to diseases.
Sanger sequencing
Sanger sequencing utilizes the chain termination method. When replicating DNA, specially designed terminating nucleotides (dideoxynucleotides, ddNTPs) are used to halt DNA synthesis at specific points. These terminating nucleotides are structurally similar to regular nucleotides (NTPs) but lack a hydroxyl group (OH) at the 3’ carbon, preventing the addition of further nucleotides.
Sanger Sequencing Procedure:
- Preparation of DNA Fragment: Prepare the DNA fragment of interest. This fragment serves as the template in the sequencing reaction.
- Preparation of Reaction Mixture: Prepare a reaction mixture containing the DNA template, primer, DNA polymerase, regular nucleotides (NTPs), and four types of dideoxynucleotides (ddNTPs, corresponding to the bases A, T, G, C). Each ddNTP is labeled with a fluorescent marker that emits light at a specific wavelength.
- DNA Synthesis and Termination: DNA polymerase synthesizes new DNA along the template, starting at the primer. When a ddNTP is incorporated by chance, DNA synthesis stops at that point, resulting in DNA fragments of varying lengths.
- Electrophoresis: The generated DNA fragments are sorted by size through electrophoresis, arranged from the shortest to the longest fragments.
- Result Interpretation: After electrophoresis, fluorescent markers are used to identify the base at the end of each fragment. This information is then used to deduce the original DNA template's sequence in reverse order.
Thus, the sequence of the entire genome is traced by combining the bases at the ends of each fragment.