Describe the steps on how the dna strand is sequenced

Answer 1

There are different methods for sequencing DNA, but one of the most commonly used techniques is the Sanger method. The following are the general steps involved in DNA sequencing using the Sanger method:

1. DNA isolation: The DNA of interest must be isolated and purified. This usually involves breaking open the cells and removing proteins, lipids, and other contaminants.

2. PCR amplification: To obtain a sufficient amount of DNA for sequencing, the target DNA is amplified using the polymerase chain reaction (PCR). PCR involves heating and cooling the DNA sample in cycles to replicate the target DNA sequence.

3. Chain termination sequencing: The Sanger method uses a modified version of the PCR reaction to sequence DNA. This method uses four fluorescently labeled nucleotides (A, C, G, T) and a primer (a short piece of DNA that binds to the target DNA sequence). In each reaction, a small amount of one of the four nucleotides is added to the reaction mixture. DNA synthesis is initiated by the primer, but when a labeled nucleotide is incorporated into the growing DNA strand, it blocks further synthesis. This termination of the chain creates fragments of varying lengths, each terminating with one of the four labeled nucleotides.

4. Capillary electrophoresis: The fragments are separated by electrophoresis in a capillary tube filled with a buffer solution and exposed to a laser. The labeled fragments migrate at different rates based on their size and charge and are detected and recorded by a computer.

5. Analysis of the sequencing data: The computer generates a chromatogram, which displays the fluorescent signals generated by the labeled nucleotides and shows the sequence of the DNA fragment. The sequence is read from the chromatogram by identifying the peaks corresponding to each nucleotide.

6. Assembly and analysis of the genome: The sequencing data is then assembled into a contiguous sequence and analyzed. This involves aligning the sequence to a reference genome, identifying variations, and annotating genes and other functional elements.