Sanger Sequencing Steps
There are three main steps to Sanger Sequencing:
The Sanger sequencing method starts with a chain termination PCR. This works just like regular PCR, but has one important difference. In contrast to standard PCR, besides normal dNTPs also a low ratio of modified nucleotides are added. These modified dNTPs are called dideoxynucleotides (ddNTPs) and have a fluorescent label also called DYe. The chain termination PCR generates DNA fragments of varying lengths, each terminated with a fluorescent labeled dideoxynucleotide.
To enable this, double stranded DNA needs to be denaturated. A primer is attached to the single stranded DNA and elongated with a mixture of nucleotides and a small quantity of chain-terminating dideoxynucleotides. No nucleotide can be added to the DNA chain once a dideoxynucleotide has been incorporated, due to the lack of the 3`OH group required for bond formation of the phosphodiester. Our recommended product is the CleanPCR.
- Size separation by Gel Electrophoresis
In the second step of the Sanger sequencing method, DNA sequences are separated based on their fragment length with capillary gel electrophoresis. Samples are loaded onto a denaturating gel matrix, and an electric current is applied. Based on their negative charge, the DNA sequencing fragments will be pulled toward the positive electrode on the opposite side of the gel. Shorter fragments experience less friction and therefor move faster. In result, the smaller fragments will be fed first into the third step of the Sanger sequencing method.
- Gel Analysis & Determination of DNA Sequence
In the early days, the ddNTP’s were radioactively labelled, allowing the detection of the final nucleotide. Modern versions use ddNTP’s which are fluorescently labelled. To determine the DNA sequence, a laser excites the fluorescent label on the dideoxynucleotide at the end of each sequence once it passes the detection window. The fluorescent label will excite a fluorescent signal, registered by a sensitive CCD camera and translated into a ‘peak’. Each of the four dideoxynucleotides is tagged with a different fluorescent label, resulting a different signal color.
Sample data is displayed as an electropherogram, a sequence of peaks in the four colors ordened by fragment size. Each color represents the base called for that peak, showing the actual DNA sequence.
Advantages of Sanger Sequencing
And then there was Next-generation sequencing (NGS)… With the arrival of this new techniques, the amount of performed Sanger sequencing analyses is slowly decreasing. Even though NGS (we would recommend the CleanNGS) has some major advantages compared to sanger sequencing, in some cases it is still worthy to perform a sanger sequencing analysis. Sanger sequencing is a fast and cost-effective method, especially for low numbers of targets. The process is simple and easy-to-use and in many cases used to confirm NGS data. We would also like to recommend the CleanDTR.