What is the primer designed for in the Sanger Sequencing Method? Also, discuss the details of the sequencing method.
The primer is designed to be complementary to the vector sequence. It provides a 3 inch hydroxyl group to which DNA polymerase can add additional nucleotides.Method steps:-The method is carried out in four test tubes. (ddTTP, ddCTP, ddATP, ddGTP). -All test tubes have the same template DNA.-Once a ddNTP is incorporated in the reaction, DNA synthesis is terminated due to the absence of the 3 inch hydroxyl group being replaced with a hydrogen. -As a result, the chain is terminated in that step.
In the Sanger sequencing method, the primer is designed to initiate DNA synthesis at a specific region of the target DNA sequence. The primer is a short DNA molecule that is complementary to the DNA sequence that is being amplified in the PCR reaction. The primer anneals to the DNA template strand and serves as the starting point for the DNA polymerase to add nucleotides and synthesize new DNA strands.
The Sanger sequencing method, also known as the chain termination method, is a technique used for DNA sequencing. It is a method of separating DNA fragments based on their length and reading the sequence of nucleotides in each fragment. The method is based on the incorporation of modified dideoxynucleotides during DNA synthesis, which results in the termination of DNA synthesis at specific bases.
The Sanger sequencing method begins with a PCR reaction that amplifies the target DNA region. The PCR reaction includes the template DNA, primers, DNA polymerase, and the four deoxyribonucleoside triphosphates (dNTPs). The DNA polymerase adds the dNTPs to the growing DNA strand to synthesize new DNA strands.
In addition to the dNTPs, the PCR reaction includes a small amount of each of the four modified dideoxynucleotides (ddNTPs), which act as chain terminators. The ddNTPs lack the 3′-hydroxyl group needed for DNA synthesis to continue, so incorporation of a ddNTP into the DNA strand terminates the DNA synthesis.
The result is a series of DNA fragments of varying lengths, each terminating at a different base, which can be separated by gel electrophoresis and visualized using fluorescence or autoradiography. The sequence of the DNA fragments can be determined by reading the sequence of bases from the smallest to the largest fragment.
Overall, the Sanger sequencing method is a widely used method for DNA sequencing, which has enabled researchers to sequence the genomes of many organisms and to identify mutations associated with genetic diseases.
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