DNA Sequencing is one of the cornerstone technologies of biotechnology and bioprocessing. It is the method of working out the sequence of DNA with a view to cloning, to understanding 3D structure etc. By using the correct analytical equipment sequencing of short of pieces of DNA is very straightforward. Sanger’s method is still applicable today.
What is Sequencing?
DNA sequencing is the process of determining the sequence of nucleotide bases, adenine (A), thymidine (T), cytosine (C) and guanine (G) in a piece of DNA.
To sequence an entire genome which is all of the organism’s DNA is extremely complex. It requires breaking genomic DNA into many smaller pieces, sequencing each of those pieces, assembling the sequences into a single long consensus. New methods have been developed over the years which are now much quicker and less expensive. Even so without those pioneering early methods we would not understand how genes are constructed. It has also led to the development of the Human genome project.
The Context For Sanger’s Sequencing Method: Chain termination.
The DNA sequencing method developed by Fred Sanger is the basis of the automated ‘cycle’ of sequencing reactions we use today. In the 1980s there were a couple of developments in molecular biology that showed scientists working in genetic engineering that it would be feasible to clone an entire genome.
The first technique was the polymerase chain reaction (PCR) which meant that many copies of a DNA sequence could be quickly and accurately produced. The second technique was the automated method of DNA sequencing which builds on the chemistry behind PCR and Fred Sanger’s sequencing process that he published in 1977. The commercial system was developed and sold by Applied Biosystems and it still remains a viable commercial sequencing method into the 2020s.
The method is ideal for relatively short pieces of DNA which are about 900 base pairs in length. It is the approach used in the Human Genome Project where small sections of DNA have been routinely analysed. To construct a larger sequence, fragments of DNA are aligned as overlapping portions. The sequences of larger sections of DNA are built up and eventually the entire chromosome is sequenced.
What Is Needed For Sanger Sequencing
Arthur Kornberg first established the principles of DNA regulation and Fred Sanger built his method upon that. A new DNA strand is synthesized using an existing strand as the template. The Sanger sequencing method relies on making copies of a target DNA region. The ingredients are the same as those required for DNA replication in all organisms and for polymerase chain reactions. What is required is:
- A DNA polymerase enzyme
- a primer which is just a short piece of single-strand DNA that binds to the template DNA and serves as the starter or initiator for the polymerase.
- The four DNA nucleotides denoted as dATP, dTTP, dCTP, dGTP.
- A piece of template DNA to be sequenced.
The key unique ingredients however which the Sanger method relies on are:-
- dideoxy or chain-terminating versions of all the four nucleotides. These are ddATP, ddTTP, ddCTP and ddGTP. Each one is labelled with a different colour dye or as a radioactive label
The dideoxynucleotides are very similar to the deoxynucleotides save for one change in the chemical structure. The dideoxynucleotides lack a hydroxyl group on the 3′ carbon of the sugar ring. In a ‘normal’ or typical nucleotide, the 3′ hydroxyl group is needed as the group used by the DNA polymerase to attach a new nucleotide and so keep on extending that sequence of DNA.
The 5′ carbon of the next added deoxynucleotide (dNTP) is joined to the 3′ carbon at the end of the chain. The hydroxyl groups in each position form ester linkages with a central phosphate. using this sequence of events, the nucleotide chain elongates. The link is usually termed the phosphodiester link.
Sanger’s method means that as with a deoxynucleotide, a ddNTP is added to the chain by forming a phosphodiester linkage at its 5′ end. In this case, the ddNTP lacks the key 3′ hydroxyl group required to form the next phosphodiester link with the next incoming nucleotide. The addition of ddNTP stops the elongation.
Methodology Of Sequencing
The DNA is sequenced by doing 4 separate reactions. Each one provides sequence information for each of the nucleotides. Each reaction contains the template DNA, a short primer of about 20 nucleotides, DNA polymerase and the four dNTP’s one of which is radioactively labeled.
One type of ddNTP is added to each reaction mixture meaning you have 4 different reactions to be monitored.
The mixture is first heated to denature the template DNA which means separating the strands. It is cooled so that the primer can bind to the single-stranded template.
The DNA polymerase does not distinguish between the dNTPs or the ddNTPs. Every time a ddNTP is incorporated, synthesis is terminated and a DNA strand is generated. For example, if ddATP was paired with thymine as the corresponding base pair. The dATP is the radioactive trace but has no effect on elongation. Because billions of DNA molecules are present, the elongation reaction is terminated at any adenine position. This produces DNA strands of different lengths. The same is true for the other three terminator reactions.
Each reaction mixture os loaded into a separate lane on an SDS-polyacrylamide gel containing urea. This compound prevents DNA strands from renaturing during electrophoresis.
Ionized phosphates give the DNA molecule a negative charge, so DNA migrates towards the positive pole of an electric field. The movement of DNA molecules through the polyacrylamide gel is size dependent.
Over the course of electrophoresis, the shorter DNA molecules move further down the gel than the larger ones. Millions of terminated DNA molecules migrate to the same place and produce a ‘band’ in the gel.
After electrophoresis the gel is sandwiched against an X-ray film. The radioactive adenine in the synthesized DNA emits beta particles which are exposed on the film. This makes a record of the position of DNA bands in the gel. The sequencing gel is then read from bottom to top. The sequence of bands in the various terminator lanes produces the sequence of nucleotides in the template DNA.
An Alternative Method Of Reading The Sequence
The process of replication is repeated in a number of cycles. By the time cycling is complete, its virtually guaranteed that a dideoxynucleotide is incorporated at every single position of the target DNA in at least one reaction. That means, the tube will contain fragments of different lengths, ending at each of the nucleotide positions in the original DNA. The ends of the fragments will be labelled with dyes that indicate their final nucleotide.
After the reaction is completed, the fragments are run through a long, thin tube containing a gel matrix in a process called capillary gel electrophoresis.
Short fragments move quickly through the pores of the gel, while long fragments move more slowly. As each fragment crosses the finishing line at the end of the tube, it is illuminated by a laser, allowing the attached dye to be detected.
From the colours of dyes registered one after another on the detector, the sequence of the original piece of DNA can be built up one nucleotide at a time. The data recorded by the detector consists of a series of peaks in fluorescence intensity. The DNA sequence is read from the peaks in the chromatogram.
The Pros And Cons of Sanger’s Method
The original Sanger sequencing method meant that highly accurate sequences for relatively long stretches of bases, up to 900 base pairs were possible. It is used now to sequence individual pieces of DNA, such as bacterial plasmids or DNA copied in the polymerase chain reaction.
The issue unfortunately with this method is its costliness and inefficient for large-scale sequence projects. Other sequencing methods have been developed which make such sequencing more economical and less time-consuming. These are used for metagenome and entire genome projects.
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