- Introduction to genetic engineering
- Intro to biotechnology
- DNA cloning and recombinant DNA
- Overview: DNA cloning
- Polymerase chain reaction (PCR)
- Polymerase chain reaction (PCR)
- Gel electrophoresis
- Gel electrophoresis
- DNA sequencing
- DNA sequencing
- Applications of DNA technologies
How the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA is determined.
- DNA sequencing is the process of determining the sequence of nucleotides (As, Ts, Cs, and Gs) in a piece of DNA.
- In Sanger sequencing, the target DNA is copied many times, making fragments of different lengths. Fluorescent “chain terminator” nucleotides mark the ends of the fragments and allow the sequence to be determined.
- Next-generation sequencing techniques are new, large-scale approaches that increase the speed and reduce the cost of DNA sequencing.
What is sequencing?
You may have heard of genomes being sequenced. For instance, the human genome was completed in 2003, after a many-year, international effort. But what does it mean to sequence a genome, or even a small fragment of DNA?
DNA sequencing is the process of determining the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA. Today, with the right equipment and materials, sequencing a short piece of DNA is relatively straightforward.
Sequencing an entire genome (all of an organism’s DNA) remains a complex task. It requires breaking the DNA of the genome into many smaller pieces, sequencing the pieces, and assembling the sequences into a single long "consensus." However, thanks to new methods that have been developed over the past two decades, genome sequencing is now much faster and less expensive than it was during the Human Genome Project.
In this article, we’ll take a look at methods used for DNA sequencing. We'll focus on one well-established method, Sanger sequencing, but we'll also discuss new ("next-generation") methods that have reduced the cost and accelerated the speed of large-scale sequencing.
Sanger sequencing: The chain termination method
Regions of DNA up to about base pairs in length are routinely sequenced using a method called Sanger sequencing or the chain termination method. Sanger sequencing was developed by the British biochemist Fred Sanger and his colleagues in 1977.
In the Human Genome Project, Sanger sequencing was used to determine the sequences of many relatively small fragments of human DNA. (These fragments weren't necessarily bp or less, but researchers were able to "walk" along each fragment using multiple rounds of Sanger sequencing.) The fragments were aligned based on overlapping portions to assemble the sequences of larger regions of DNA and, eventually, entire chromosomes.
Although genomes are now typically sequenced using other methods that are faster and less expensive, Sanger sequencing is still in wide use for the sequencing of individual pieces of DNA, such as fragments used in DNA cloning or generated through polymerase chain reaction (PCR).
Ingredients for Sanger sequencing
Sanger sequencing involves making many copies of a target DNA region. Its ingredients are similar to those needed for DNA replication in an organism, or for polymerase chain reaction (PCR), which copies DNA in vitro. They include:
- A DNA polymerase enzyme
- A primer, which is a short piece of single-stranded DNA that binds to the template DNA and acts as a "starter" for the polymerase
- The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
- The template DNA to be sequenced
However, a Sanger sequencing reaction also contains a unique ingredient:
- Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP), each labeled with a different color of dye
Dideoxy nucleotides are similar to regular, or deoxy, nucleotides, but with one key difference: they lack a hydroxyl group on the 3’ carbon of the sugar ring. In a regular nucleotide, the 3’ hydroxyl group acts as a “hook," allowing a new nucleotide to be added to an existing chain.
Once a dideoxy nucleotide has been added to the chain, there is no hydroxyl available and no further nucleotides can be added. The chain ends with the dideoxy nucleotide, which is marked with a particular color of dye depending on the base (A, T, C or G) that it carries.
Method of Sanger sequencing
The DNA sample to be sequenced is combined in a tube with primer, DNA polymerase, and DNA nucleotides (dATP, dTTP, dGTP, and dCTP). The four dye-labeled, chain-terminating dideoxy nucleotides are added as well, but in much smaller amounts than the ordinary nucleotides.
The mixture is first heated to denature the template DNA (separate the strands), then cooled so that the primer can bind to the single-stranded template. Once the primer has bound, the temperature is raised again, allowing DNA polymerase to synthesize new DNA starting from the primer. DNA polymerase will continue adding nucleotides to the chain until it happens to add a dideoxy nucleotide instead of a normal one. At that point, no further nucleotides can be added, so the strand will end with the dideoxy nucleotide.
This process is repeated in a number of cycles. By the time the cycling is complete, it’s virtually guaranteed that a dideoxy nucleotide will have been incorporated at every single position of the target DNA in at least one reaction. That is, the tube will contain fragments of different lengths, ending at each of the nucleotide positions in the original DNA (see figure below). The ends of the fragments will be labeled with dyes that indicate their final nucleotide.
After the reaction is done, 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 “finish line” at the end of the tube, it’s illuminated by a laser, allowing the attached dye to be detected.
The smallest fragment (ending just one nucleotide after the primer) crosses the finish line first, followed by the next-smallest fragment (ending two nucleotides after the primer), and so forth. Thus, from the colors 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 consist of a series of peaks in fluorescence intensity, as shown in the chromatogram above. The DNA sequence is read from the peaks in the chromatogram.
Uses and limitations
Sanger sequencing gives high-quality sequence for relatively long stretches of DNA (up to about base pairs). It's typically used to sequence individual pieces of DNA, such as bacterial plasmids or DNA copied in PCR.
However, Sanger sequencing is expensive and inefficient for larger-scale projects, such as the sequencing of an entire genome or metagenome (the “collective genome” of a microbial community). For tasks such as these, new, large-scale sequencing techniques are faster and less expensive.
The name may sound like Star Trek, but that’s really what it’s called! The most recent set of DNA sequencing technologies are collectively referred to as next-generation sequencing.
There are a variety of next-generation sequencing techniques that use different technologies. However, most share a common set of features that distinguish them from Sanger sequencing:
- Highly parallel: many sequencing reactions take place at the same time
- Micro scale: reactions are tiny and many can be done at once on a chip
- Fast: because reactions are done in parallel, results are ready much faster
- Low-cost: sequencing a genome is cheaper than with Sanger sequencing
- Shorter length: reads typically range from nucleotides in length
Conceptually, next-generation sequencing is kind of like running a very large number of tiny Sanger sequencing reactions in parallel. Thanks to this parallelization and small scale, large quantities of DNA can be sequenced much more quickly and cheaply with next-generation methods than with Sanger sequencing. For example, in 2001, the cost of sequencing a human genome was almost . In 2015, it was just !
Why does fast and inexpensive sequencing matter? The ability to routinely sequence genomes opens new possibilities for biology research and biomedical applications. For example, low-cost sequencing is a step towards personalized medicine – that is, medical treatment tailored to an individual's needs, based on the gene variants in his or her genome.
Want to join the conversation?
- This might be a bit off topic, but I am a chemistry student and I want to get a tattoo of my father DNA. Now I know the human DNA contains too many basepairs to fit as a tattoo. But if a small segment of the sequence is used, how likely is it that that sequence also belongs to a different person?(20 votes)
- Technically speaking, you could use the sequence from DNA fingerprinting (the method used to identify a person), but it would be still a lot of material for a tattoo, it's typically thirteen sequences of varying length... I think it would be pretty big tattoo.
Another option would be getting a tattoo of "DNA ladder" (the DNA fingerprinting pattern seen on electrophoresis), this is also unique for a person and DNA related. And add only two or three nice, detailed basepairs next to it.(18 votes)
- Are there two systems to sequencing?
one with light (laser) and the second with electric field?(3 votes)
- In traditional (Sanger) sequencing, both electric fields and lasers are required for slightly different, but interdependent, purposes. The electric field is applied to the DNA in the capillary tube, and it pulls the DNA pieces through in order from shortest to longest. As the pieces pass the laser (moving through because of the electric field), they are excited and detected by the detector.
Some next-generation sequencing does not use lasers at all. Instead, it uses H+ ion fluxes to determine whether bases have been added. I'm not super familiar with this technique, but you can read more in this Wikipedia article: https://en.wikipedia.org/wiki/Ion_semiconductor_sequencing.
Hope one of those answers addresses your question!(10 votes)
- Why can't the die molecule be attached to a regular nucleotide, and then the entire DNA chain could be read as a single item?(6 votes)
- you cannot read the entire dna chain as a single item, even if each base pair were to be dyed since you would be getting the four colours at once due to the small size of the molecule. you use ddntp to stop the synthesis for a strand and get fragments of all possible lengths that move in ascending order of length. this lets you to exactly know which base comes at what point.(3 votes)
- Does a 2000bp or a 500bp migrate faster throgh this agarose gell(2 votes)
- In other words, the 500bp would migrate faster.(4 votes)
- How do we know the specific primer if we already don’t know the DNA fragment sequence ?(4 votes)
- If we want to amplify a fragment of DNA, but don't know the sequence a technique known as "Ligation-mediated PCR" can be used. This technique starts by adding (ligating) the primer sequences to the ends of the DNA fragment.(3 votes)
- Why do the nucleotides used here have 3 phosphate groups instead of 1?(2 votes)
- It's to provide it with energy. The regular DNA replication also uses 3phosphate groups because by removal of each one of them, it gets enough energy to bind to the previous nucleotide.(4 votes)
- in a chromatogram why are the peaks of different heights?(3 votes)
- The height of each peak is in proportion to the amount of the particular component present in the sample mixture injected into the chromatograph.(2 votes)
- what molecule can be added to a 1st gen and second gen sequencing that will halt the reaction but will not affect a third gen sequencing?(3 votes)
- They all rely on adding nucleotides to continue the reaction.
The difference between next-generation sequencing and Sanger sequencing is that NGS allows us massive parallel sequencing.
In Sanger Sequencing, dideoxynucleotides are added to stop sequencing and help us read the products.
With NGS (Illumina in this example but the concept is the same for other NGS technologies) you prepare a target-DNA library and you load it into a flowcell in order to generate DNA clusters that are then sequenced in parallel. What is really important to understand is that each cluster is generated from a SINGLE MOLECULE of target DNA. This allows you to 'physically' separate the signal originating from each target molecule. Meaning that adding steps won't stop the process.
Actually, they work on different principles and do not know how to exactly answer your question.
- This may go a bit further than what the article specified, but here we go. In next-generation sequencing, how would you prevent reading a double nucleotide. If you add multiple dNTPs to a single cluster, and the next sequence is AA, how would you be able to tell the difference between this and just A?(3 votes)
- depending on the method you are reading it the answer may change. but let me give you one example i know
in the ones that we observe the dntp by the light if it is aa it emits more light if it is a smaller amount of light emitted. if there is aaa there is even more emitted but more than 4 aaaa is being problem that we can not observe the differences between emitions. you can watch a small video on youtube about next generation sequencing i would reccomend the one with ion torrent, which gives your answer specially(1 vote)
- How is it possible to get the sequence of the original DNA if the whole sample is probably disorganized in the mixture?(3 votes)
- there will be many fragments as you say and it is a huge confussion soup. after they read all the data together they try to match matching ones and try to fill the whole sequence by fitting all matches next to each other. assume like dna consist of numbers from 1 to 6and the sequence is 1 2 3 4 5 6
when you pcr this dna and cut in the fragments you will have results like 12 234 345 456 23 34 56 123 456 234 123 45 34 when you use a computer to match them it will find it as 123456 (think numbers as serries of actg)(1 vote)