Anyone who is involved in DNA sequencing in one form or fashion knows there are multiple ways to skin a cat: Sanger-based, next generation (NGS) and of course the new ion torrent sequencing technology. Which technology you use is usually dependent on the questions you’re trying to answer – and how fat your wallet is.
When I first read about pyrosequencing many years ago, I had this vision of scary looking scientists with spiky hair, thick black goggles and long black rubber gloves pouring fiery green ooze into a sequencing plate. Okay, not quite that scary. In 1996, two scientists working at Stockholm’s Royal Institute of Technology developed a novel way to sequence DNA in real-time. This new sequence-by-addition or “synthesis” method is called “pyrosequencing.” The term “pyro” refers to the dependence of measuring the release of pyrophosphate (PPi) during nucleotide incorporation.
Essentially, the process begins with the incorporation of nucleotides one at a time as a complementary, single strand to the ssDNA template. As the nucleotides are added, light from the release of PPi is emitted and measured (usually via a CCD camera). The user controls at which point each A, T, C or G is sequentially dispensed, allowing easy tracking of the desired sequence loci. Repeat nucleotide incorporation (A-A, G-G-G, T-T-T-T etc) will give a stronger light emission signal relative to the signal given by incorporation of a single copy.
Before we take you through an actual procedure, it’s important to point out a couple of quick notes. First, using higher amplification cycles (40-50) helps to exhaust the biotinylated PCR product. This ensures sufficient product yield and serves to avoid artifacts appearing later in your sequencing reaction and the cleaner the PCR product, the better. Second, pyrosequencing can generally detect cDNA in the 1-10 picomol (pmol) range so it’s wisest to start with a lower concentration of primers (~ 10pmol versus 40 pmol).
1) Using a primer pair, one being biotinylated, amplify the region of interest (usually 30 – 200 bp).
2) Verify PCR product by resolving on agarose gel (most common).
3) Using a mastermix strategy, bind your biotinylated PCR product to stretavidin beads.
4) Using a handheld vacuum plate, isolate the bead-amplicon products.
a. Wash in 70% ethanol to remove unwanted PPI.
b. Denature the dsDNA strand to just the biotinylated strand using sodium hydroxide (NaOH).
5) Annealing of the biotinylated amplicon to the sequencing primer is through a series of heat and cool steps.
6) A cartridge is prepared that will hold the chemistry enzymes and substrate as well as the 4 individual dNTPs.
The Chemistry of Pyrosequencing
Let’s take a closer look at what’s going on inside during the sequencing reaction. After primer hybridization to the template, the first sequential addition of nucleotides are dispensed one at a time in the presence of a DNA polymerase. If the first template nucleotide is an A, a T will be incorporated into the new strand. This releases PPI stoichiometrically. One of the enzymes in the mix, ATP sulfurylase, converts the newly-released PPI into ATP via the presence of the substrate, adenosine 5’ phosphosulfate. These new ATP molecules will drive the reaction of another substrate, luciferin into oxyluciferin via another enzyme, luciferase. The oxyluciferin is the visible light generated which also happens to be directly proportional to the ATP present. Finally, all the nucleotides and ATP not used during each sequential dispensation are “chewed up” by a clever little enzyme called apyrase. The growing new DNA strand is tracked by a computer program both with a qualitative and quantitative representation of the new sequence. Generally, a 96 well plate containing 60+bp amplicons can be read and reported in about an hour.
Advantages and Limitations of Pyrosequencing
Pyrosequencing has been around for a little over 15 years now and the technology has become the standard for which large genomes are easily sequenced at a fraction of the time and cost of Sanger sequencing methods. Depending on the platform used, pyrosequencing does have some limitations. As mentioned above, sequencing short loci of interest can be accomplished quickly, but target sequences of >160 bp are the limits of certain licensed formats, like Qiagen’s Pyromark ID 96 and Q24 Advanced systems. These systems are actually replacing obsolete real-time PCR equipment as the costs are about the same, but the product is far more useful. Larger systems like 454 Life Sciences’ array-based sequencer are used for large scale DNA sequencing.
If your aim is not to sequence full genomes, pyrosequencing affords a multitude of applications from Qiagen’s SNP detection to CpG methylation. We’ll discuss the applications available in the next article. In the meantime, if you are currently using pyrosequencing, what are YOUR applications and what do you like/dislike about the technology?