terminator sequencing of a dna
Terminator sequencing, also known as fluorescent dye terminator sequencing, is a method used to sequence DNA. The DNA to be sequenced is first amplified using the polymerase chain reaction (PCR) to amplify it. The DNA is then mixed with short fragments of DNA known as primers, which bind to the beginning of the DNA sequence being analyzed.
Next, DNA polymerase is used to add fluorescent dyes to the DNA as the sequencing proceeds. Each of the four DNA nucleotides (adenine, thymine, cytosine, and guanine) is labeled with a different color of fluorescent dye. The sequencing reaction is then stopped using a chemical termination process.
The DNA fragments are separated by size using gel electrophoresis, with smaller fragments migrating further down the gel than longer ones. The fluorescent dyes are excited using a laser, and the emitted light is captured by a detector. The sequence of the DNA can then be read by analyzing the colors of the fluorescent dyes that are detected at each step.
Terminator sequencing is a highly accurate and efficient method for sequencing DNA, and is commonly used in research, clinical, and forensic applications.
Terminator sequencing, also known as Sanger sequencing or chain termination sequencing, is a method used to determine the DNA sequence of a particular segment. Here's a step-by-step explanation of the process:
1. DNA Extraction: Obtain the DNA sample containing the segment of interest. This can be done by isolating DNA from cells or tissues using various methods.
2. PCR Amplification: Perform a polymerase chain reaction (PCR) to selectively amplify the specific DNA segment of interest. This is done using two primers that flank the region to be sequenced.
3. Cycle Sequencing Reaction: Set up a series of four separate sequencing reactions, each containing the DNA template, a sequencing primer, DNA polymerase, deoxynucleotides (dNTPs; A, T, G, C), and modified dideoxynucleotides (ddNTPs; labeled with different fluorescent dyes and lacking a 3' hydroxyl group).
4. Chain Termination: During the sequencing reaction, DNA polymerase incorporates the standard dNTPs into the growing DNA strands until it encounters a ddNTP. The lack of a 3' hydroxyl group in ddNTPs prevents further chain elongation.
5. Gel Electrophoresis: Separate the terminated DNA fragments by size using gel electrophoresis. The gel contains polyacrylamide or agarose matrix, and an electric field is applied to move the DNA molecules through the gel.
6. Data Acquisition: Perform fluorescence detection on the gel to determine the base identity of each terminated fragment. The different labeled ddNTPs emit fluorescence at different wavelengths, indicating the base present at each position.
7. Data Analysis: Analyze the fluorescence signals to determine the sequence of the DNA segment. The sequence can be inferred by identifying the color and intensity of the peaks corresponding to each base in the electropherogram.
8. Assembly and Verification: If the DNA sequence of interest is longer than the read length of a single sequencing reaction, multiple reactions covering overlapping regions need to be performed. The obtained sequences are then aligned and assembled into a contiguous sequence.
Overall, Terminator sequencing allows for the determination of the DNA sequence in a stepwise manner using chain termination principles and detection of fluorescently labeled ddNTPs.