Describe how a dna strand is sequenced with the sanger sequencing method
The Sanger sequencing method is a technique that allows the determination of the sequence of DNA nucleotides within a strand of DNA. This method is based on the incorporation of dideoxynucleotides, which are modified nucleotides that lack the 3' hydroxyl group necessary for the formation of phosphodiester bonds with the next nucleotide.
The process of Sanger sequencing begins with the preparation of a DNA template strand that is to be sequenced. This template strand is then mixed with a DNA polymerase enzyme, four types of deoxynucleotides (dATP, dCTP, dGTP, and dTTP), and a small amount of one of the four dideoxynucleotides (ddATP, ddCTP, ddGTP, or ddTTP). The dideoxynucleotide is labeled with a fluorescent dye to distinguish it from the deoxynucleotides.
The polymerase enzyme will incorporate the deoxynucleotides into a newly synthesized strand of DNA until it incorporates the modified dideoxynucleotide, which lacks the 3' hydroxyl group required for forming the next phosphodiester bond. Due to this deficiency, the incorporation of the dideoxynucleotide terminates strand elongation.
This reaction generates fragments of varying lengths, each ending with a dideoxynucleotide at its 3' end. These fragments are then separated by size using a gel electrophoresis apparatus based on their changes in electrophoretic mobility. The resulting bands of fragments are then analyzed using a laser scanner or fluorescence detector that identifies the color of the attached dye.
This technique produces a series of fluorescent peaks corresponding to the sequence of nucleotides within the original DNA strand. The order of fluorescent peaks indicates the sequence of the DNA strand, with each peak representing a specific nucleotide in the sequence, and the length of the fragments representing the location of each nucleotide within the original strand.
The Sanger sequencing method, also known as dideoxy sequencing or chain-termination sequencing, is a widely used technique to determine the order of nucleotides (A, T, C, G) in a DNA strand. Here is a step-by-step description of the Sanger sequencing process:
1. DNA Preparation: The first step involves isolating the DNA of interest and amplifying it using PCR (Polymerase Chain Reaction), creating multiple copies. This provides enough DNA to work with during the sequencing process.
2. DNA Primers: DNA primers are short sequences of DNA that bind to the specific regions of the DNA template to be sequenced. These primers are designed to flank the target region, which ensures that the entire region of interest is amplified.
3. DNA Synthesis: Once the DNA template and primers are prepared, DNA synthesis is carried out. This process involves adding the four nucleotide building blocks (A, T, C, G), along with a small amount of modified nucleotides called dideoxynucleotides (ddNTPs) into separate reaction tubes. The ddNTPs are labeled with fluorescent dyes of different colors (e.g., ddATP labeled with green dye).
4. Chain Termination: During DNA synthesis, when a regular nucleotide (A, T, C, G) incorporates into the growing DNA strand, it extends the DNA chain. However, when a ddNTP is incorporated, the chain elongation terminates because ddNTPs lack a 3'-OH group essential for further chain elongation.
5. Gel Electrophoresis: After the DNA synthesis reaction, the resulting mixture contains a collection of DNA fragments of different lengths. These fragments need to be separated based on their length to determine the order of nucleotides. The mixture is loaded onto an agarose or polyacrylamide gel, and an electric current is applied. Smaller DNA fragments move more quickly through the gel than longer ones.
6. Visualization and Data Interpretation: Once the fragments have separated based on size, the gel is exposed to ultraviolet (UV) light or laser, which excites the fluorescent dyes present on the ddNTPs. The dye emits light of different colors corresponding to each nucleotide. A camera or detector captures the emitted light signals.
7. Data Analysis: The emitted light signals are converted into a series of digitized peaks (called electropherogram), where each peak represents a specific nucleotide at a particular position in the DNA strand. These peaks are then analyzed using specialized software that identifies the overlapping sequences, assigns base calls (A, T, C, G), and generates the final DNA sequence.
8. Sequence Assembly: After obtaining the nucleotide sequence from multiple readouts (usually multiple sequencing reactions are conducted), the DNA sequence is assembled by overlapping the regions with matching sequences. This step helps to account for any sequencing errors or discrepancies.
By following these steps, the Sanger sequencing method allows scientists to determine the sequence of DNA strands, providing valuable information for research, diagnostics, and various applications in the field of genetics.