Title: Analyzing Sequencing Data
Course: BTEC-1100-301-302-303-F21 > Section: Module 4 - Applied Molecular Biology

Your miniprep DNA from the last activity will be sent off to an offsite sequencing facility to determine if the bglA gene was correctly cloned into the pLATE31 plasmid.

DNA Sequencing

A basic sequencing technique is the chain termination method, also known as the dideoxy method or the Sanger DNA sequencing method, developed by Frederick Sanger in 1972. The chain termination method involves DNA replication of a single-stranded template with the use of a DNA primer to initiate synthesis of a complementary strand, DNA polymerase, a mix of the 4 regular deoxynucleotide (dNTP) monomers, and a small proportion of dideoxynucleotides (ddNTPs), each labeled with a molecular beacon. The ddNTPs are monomers missing a hydroxyl group (–OH) at the site at which another nucleotide usually attaches to form a chain Every time a ddNTP is randomly incorporated into the growing complementary strand, it terminates the process of DNA replication for that particular strand. This results in multiple short strands of replicated DNA that are each terminated at a different point during replication. When the reaction mixture is subjected to gel electrophoresis, the multiple newly replicated DNA strands form a ladder of differing sizes. Because the ddNTPs are labeled, each band on the gel reflects the size of the DNA strand when the ddNTP terminated the reaction.

In Sanger’s day, four reactions were set up for each DNA molecule being sequenced, each reaction containing only one of the four possible ddNTPs. Each ddNTP was labeled with a radioactive phosphorus molecule. The products of the four reactions were then run in separate lanes side by side on long, narrow PAGE gels, and the bands of varying lengths were detected by autoradiography.

Today, this process has been simplified with the use of ddNTPs, each labeled with a different colored fluorescent dye or fluorochrome, in one sequencing reaction containing all four possible ddNTPs for each DNA molecule being sequenced. These fluorochromes are detected by fluorescence spectroscopy. Determining the fluorescence color of each band as it passes by the detector produces the nucleotide sequence of the template strand.

A dideoxynucleotide is similar in structure to a deoxynucleotide but is missing the 3ʹ hydroxyl group (indicated by the shaded box).  Then a dideoxynucleotide is incorporated into a DNA strand, DNA synthesis stops.

Frederick Sanger’s dideoxy chain termination method is illustrated, using ddNTPs tagged with fluorochromes. Using ddNTPs, a mixture of DNA fragments of every possible size, varying in length by only one nucleotide, can be generated. The DNA is separated by size, and each band can be detected with a fluorescence detector.

This diagram summarizes the Sanger sequencing method using fluorochromelabeled ddNTPs and capillary gel electrophoresis.

Some videos on sequencing can be found here: Thermofisher

And here:

Important: When the results for your miniprep are available your instructor will show you how to use the NCBI database to determine if your gene was successfully cloned.