# DNA-Sequencing (detection of the change of pH-value) after emulsion PCR

I have troubles with understanding the exact order of the steps for DNA-sequencing after the Emulsion PCR (EmPCR). The EmPCR fills each bead with the same DNA strand (copies it $$n$$ times, till the bead is entirely filled up). But how is then the sequencing done with these beads?

The sequencing is described as follows (in easy words): Each nucleotide is provided to the DNA strand. If a nucleotide is accepted, the $$\ce{H+}$$ concentration rises and can be detected. Hence, the complementary nucleotide was present at the DNA strand.

But to be able to attach a nucleotide to the DNA, it has to be broken up. But the EmPCR already filled up every copied DNA strand. Where is it then broken up again?

• May I suggest migration to Biology.SE? Please use the biotechnology and/or biochemistry tag there if you do migrate. – William R. Ebenezer Jun 9 at 17:30
• Are you using Roche-454 pyrosequencing machine? – Mathew Mahindaratne Jun 9 at 22:31

In molecular biology, it is a very important that DNA should be replicated in order to facilitate sequencing. Basically, this is a very important concept that DNA should be replicated, simply in order to amplify signals. No matter the method of sequencing, without a proper amount of amplification, it's near impossible to detect each base call (signal). All Next-Generation Sequencing (NGS) techniques replicate DNA before sequencing is done.

Emulsion PCR is a PCR variation that some NGS technologies (e.g., NGS flatforms: Roche/454’s GS FLX, Titanium; Life/APG’s SOLiD 3; and Polonator G.007) use to replicate DNA sequences. It is conducted on a bead surface within tiny water bubbles floating on an oil solution.

1. Fragmentation of DNA: The library is first fragmented either by sonication (high sound energy) or nebulization (forces DNA through a small hole) to fragments ranging from 300 to 800 base pairs.
2. Adapters are ligated: Adapters are then ligated onto the DNA fragments. These allow the strands to bind to the emulsion beads.
3. Denature to single strands: The double stranded DNA's with adapters are then denatured by heating the DNA up to $$\pu{95 °C}$$. Denaturing DNA simply means to go from double stranded DNA (dsDNA) to two single strands (ssDNA) - the hydrogen bonds keeping the two together are broken.
4. Formation of clonal bead populations: Each bead coated with streptavidin (e.g., Streptavidin-coupled Dynabeads), which is resistant to organic solvents, denaturants, detergents, proteolytic enzymes and extremes of temperature and $$\mathrm{pH}$$. Over a billion beads are used with a primer that matches the adapters attached earlier. The ssDNA is then attached to these beads. Each bead is emulsified in a water-in-oil droplet with PCR reagents (DNA polymerase, primers, buffers, dNTPs):

5. Emulsion PCR (emPCR) amplifies DNA strands on beads: Within these droplets, PCR is conducted. This involves the steps Denaturation, Annealing, Elongation. Firstly, the strand is elongated with DNA polymerase and dNTPs. Then the double-strand is denatured, allowing for the strand to ligate to another site on the surface of the bead. Eventually, 1 million copies of the target is amplified on the surface of each bead. The water-in-oil droplet is approximately 1-um. Follow the figure to see how each bead is able to replicate DNA on its surface.

1. Emulsion Breaking: After the DNA strands are amplified, the emulsion from the preceding step is broken using isopropanol and detergent buffer. The solution is then vortexed, centrifuged, and magnetically separated. The resulting solution is a suspension of empty, clonal and non-clonal beads, which will be filtered in the next step.
2. Bead enrichment: After PCR is conducted, you are left with a mixture of some beads that have amplified DNA attached on its surface, and some that do not. We may take out the enriched beads by attaching streptavidin coated magnetic enrichment bead. With a magnet, we can then pull out the beads with amplified DNA. There are other methods of bead enrichment that include using larger beads that are able to bind to beads with amplified DNA. After centrifugation, the beads with amplified DNA and without can then be separated.

3. Bead Capping: Attach a capping oligonucleotide to the 3' end of both unextended forward EmPCR primers and the RDV segment of template DNA. This helps in cover-slip arraying, which is used to polony sequencing, and prevents fluorescent probes from ligating to the ends.

4. Result: The beads with amplified sequences are then placed on a slide and are sequenced. Due to their high density of the same DNA molecule, the signal is amplified, allowing computers to read the sequencing data.

For example, the method used by the Roche/454 sequencer to amplify single-stranded DNA copies from a fragment library on agarose beads. A mixture of DNA fragments with agarose beads containing complementary oligonucleotides to the adapters at the fragment ends are mixed in an approximately 1:1 ratio. The mixture is encapsulated by vigorous vortexing into aqueous micelles that contain PCR reactants surrounded by oil, and pipetted into a 96-well microtiter plate for PCR amplification. The resulting beads are decorated with approximately 1 million copies of the original single-stranded fragment, which provides sufficient signal strength during the pyrosequencing reaction that follows to detect and record nucleotide incorporation events. sstDNA, single-stranded template DNA (Ref.1):

Keep in mind that in pyrosequencing method, each incorporation of a nucleotide by DNA polymerase results in the release of pyrophosphate, which initiates a series of downstream reactions that ultimately produce light by the firefly enzyme luciferase. The amount of light produced is proportional to the number of nucleotides incorporated (up to the point of detector saturation). Above diagram shows an example of four-color mapping sequence. For additional info, should read all of Ref.1-3.

References:

1. Elaine R. Mardis, “Next-Generation DNA Sequencing Methods,” Annu. Rev. Genomics Hum. Genet. 2008, 9, 387–402 (doi: 10.1146/annurev.genom.9.081307.164359).
2. R. Kanagal-Shamanna, “Emulsion PCR: Techniques and Applications,” Methods in Molecular Biology, Volume 1392: Clinical Applications of PCR; R. Luthra, R. Singh, K. Patel, Eds.; Humana Press: New York, NY, 2016, pp. 33–42 (https://doi.org/10.1007/978-1-4939-3360-0_4).
3. Michael L. Metzker, “Sequencing technologies —the next generation,” Nature Reviews Genetics 2010, 11, 31–46 (doi: 10.1146/annurev.genom.9.081307.164359).
• Does the "Emulsion Breaking" mean, that the DNA strands on the Beads are again broken that only the single strand DNA remains on the Beads? – vogs Jun 10 at 9:17
• No, it's double strand DNA, which you are targeting. Based on method of breakup, only streptavidin-coupled beads or those with PI-adapter (linked to streptavidin by biotin link. Your targeted DNA would remain in the solution and ready to sequence after specific separation (or go to DNA library). – Mathew Mahindaratne Jun 10 at 15:22
• @vogs: Also, you must read those articles and get some understanding. And, it would have been very helpful if you had provided your protocol. – Mathew Mahindaratne Jun 10 at 15:25
• I'm trying. But somehow every explanation of the sequencing (let's take the Roche/454) starts with ssDNA on it's strands but the emPCR produces dsDNA on the Beads. – vogs Jun 10 at 17:26
• That's correct. – Mathew Mahindaratne Jun 10 at 17:47

Before the sequencing starts, the DNA on the bead is denatured (the strand that is covalently boudn remains, the other is washed away). Polymerase and primer are added. Then, the four nucleotides are offered one by one, leading to extension or not. The pH change not only shows whether a nucleotide is attached, but also how many (in case of runs of a given nucleotide in the template strand. This picture illustrates the process:

• Thanks for the answer. This means, that the denaturation is done in the emPCR step (several times) as well as before the sequencing starts? I am always missing this part in the explanations. E.g. when looking here on slide 16 the Bed is filled with full DNA strands but in slide 25 a single strand DNA is used for sequencing. But no one explains the step in between. – vogs Jun 10 at 9:14
• @vogs I guess so. In the classic PCR protocol, denaturation is critical because it supports the other two steps - primer annealing and primer elongation. Without denaturation (or melting), you would not get exponential amplification. If you like the answer, you can upvote it or accept it. – Karsten Theis Jun 10 at 18:09