02. Sanger sequencing chain-termination method
Sanger sequencing was developed by Frederick Sanger and his colleagues in 1977. The development of this technique won Sanger the Nobel Prize in Chemistry in 1980.
From the 80's to the mid-2000's, Sanger sequencing dominated the DNA sequencing platform, bringing successful completion of the Human Genome Project (HGP) in 2003. Although this technique has been replaced by next generation sequencing methods, it is still used today for smaller-scale projects.
What is dideoxynucleotide?
A dideoxynucleotide (ddNTP) is an artifical molecule that lacks a hydroxyl group at both the 2' and 3' carbons of the sugar moiety. Compare this to a regular deoxynucleotide triphosphate (dNTP), which has the hydroxyl group on the 3' sugar.
The main purpose of the 3'-OH group is that it is used to form a phosphodiester bond between two nucleotides - this is what allows for a DNA strand to elongate.
DNA elongation cannot occur with ddNTPs
During DNA replication, an incoming nucleoside triphosphate is linked by its 5' α-phosphate group to the 3' hydroxyl group of the last nucleotide of the growing chain. With ddNTP, where there is no 3' hydroxyl group, this reaction cannot take place, so elongation is terminated.
Here is an image of how DNA elongation regularly occurs (with dNTP instead of ddNTP).
Now that we have seen the chemistry behind the ddNTP, let's look at how Sanger Sequencing works.
Procedure
There are three main steps in Sanger Sequencing, as outlined below.
1) Clone DNA strands into a vector
The first step is to fragment the DNA and clone the fragments into vectors.
2) Attach primer
The second step is to anneal a synthetic oligonucleotide with length 17 to 24-mer. (An oligonucleotide is just a fancy name for a short strand of DNA). The oligonucleotide acts as a binding site for a primer and provides a 3' hydroxyl group, which is necessary to initiate DNA synthesis.
In order to recognize the sequence and identify precisely the first nucleotide of the target DNA, the primer is usually positioned 10 to 20 nucleotides away from the target DNA.
2) Add four dNTPs + 1 ddNTP
Four different reaction vials are made, each with the four standard dNTP's, and DNA polymerases.
The difference among the vials are the type of ddNTPs. Each vial will have 1 ddNTP per 100 dNTP.
After DNA synthesis occurs, each reaction vial will have a unique set of single-stranded DNA molecules of varying lengths. However, all DNA molecules will have the same primer sequence at its 5' end.
The resulting DNA fragments are then denatured by heat since base-paired loops of ssDNA may cause difficulty in resolving bands when running a gel. Additionally, one may add formamide to prevent base pairing.
3) Find sequence by gel electrophoresis
Now that we have varying sequences, we need to line them up according to size to determine the sequence.
Radiography
Here, the ddNTPs would have to be radioactively or fluorescently labeled beforehand for automated sequencing machines. The DNA strands are then separated using gel electrophoresis, then read from top to bottom (3' to 5') to obtain the sequence.
Dye-terminating sequencing
We could have fluorescently labeled each ddNTP to use dye-terminating sequencing instead. This causes each of the four ddNTPs to emit light at different wavelengths. Here, we capillary electrophoresis, with a single lane to capture the nucleotide sequence.
Pros
- Not as toxic and less radioactivity than Maxam and Gilbert method.
- Easier to automate - Leroy Hood and coworkers used fluorescently labeled ddNTPs and primers for the first high-throughput DNA sequencing machine. This lowered the cost from $100 million to $10,000 USD in 2011.
- Highly accurate long sequence reads of about 700 base pairs.
- Easier to get started. The kits that are commercially available contain reagents necessary for sequencing - pre-aliquoted and ready to use.
Cons
- Poor quality in the first 15-40 bases of the sequence. This is due to primer binding and deteriorating quality of sequencing traces after 700-900 bases.
- Time consuming, especially due to requirement for electrophoretic separation of fragments. Expensive due to relatively large volumes of chemicals that are used.
- DNA fragments cloned before sequencinging - read may include parts of the cloning vector.
- Only short 300-1000 nucleotides long DNA fragments in a single reaction. Problem with reading strands longer is the insufficient power of separation for resolving large DNA fragments that differ in length by only one nucleotide.
- To elongate these reads, we may use a technique known as Primer Walking, which we will see in the next lesson.
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