Everything You Need to Know about Sanger Sequencing


Posted August 3, 2022 by henryjosephbio

If you are looking for an online portal for sanger sequencing services, try out Bio Basic. Get affordable pricing, free delivery, and the best quality service.

 
Sanger sequencing is also known as the chain termination method. Sanger sequencing can be performed manually or more commonly, in an automated fashion via a sequencing machine. Each method follows three basic steps.

A unique kind of PCR termed chain-termination PCR uses the target DNA sequence as a template. While chain-termination PCR functions similarly to normal PCR, it differs significantly in that modified nucleotides (dNTPs) known as deoxyribonucleotides are added (ddNTPs). By catalyzing the creation of a phosphodiester bond between the free 3'-OH group of the last nucleotide and the 5'-phosphate of the following nucleotide, DNA polymerase adds dNTPs to a developing DNA strand during the extension stage of conventional PCR.

The next stage involves separating the chain-terminated oligonucleotides by size using gel electrophoresis. The oligonucleotides will be drawn toward the positive electrode on the other side of the gel in gel electrophoresis because DNA is negatively charged. DNA samples are placed into one end of a gel matrix and an electric current is applied. Because each DNA fragment has the same charge per unit of mass, only size will affect how quickly oligonucleotides move.

The final step consists of simply reading the gel to ascertain the input DNA sequence. Each terminal ddNTP will correspond to a specific nucleotide in the original sequence because DNA polymerase only creates DNA in the 5' to 3' direction starting at a provided primer (for example, the shortest fragment must terminate at the first nucleotide from the 5' end, the second-shortest fragment must terminate at the second nucleotide from the 5' end, etc.). Therefore, we may ascertain the 5' to 3' sequencing of the original DNA strand by analyzing the gel bands from smallest to largest.

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Last Updated August 3, 2022