Apr 122017
 

Once we realised that most of our plate of Schistidium ITS2 amplifications had been successful, it was an easy decision to process them all for DNA sequencing. If a higher proportion had failed, we would have had to “cherry pick”, selecting and transferring the successful reactions into new tubes. Every sequencing reaction has a cost, and so deliberately sending a lot of failed reactions through the process, knowing that they won’t generate DNA sequences, is worth avoiding. However, transferring the reactions into new tubes is a meticulous job. In a plate of reactions, there are 96 wells arranged in 12 columns (1-12) and eight rows (A-H).  The first sample is at 1A, then 1B, 1C, 1D, 1E, 1F, 1G, 1H, 2A… all the way to 12H. Imagine if the first failed sample is at 1B and the second at 1G – then we’d transfer 1A to 1A, 1C to 1B, 1D to 1C, 1E to 1D, 1F to 1E, 1H to 1F, 2A to 1G, etc… it’s easy to see that any interruptions can be fatal (this sort of task is why people in the lab sometimes have a “Do Not Disturb” sign stuck on the back of their lab coats). Thus, with only a few failures, we’re better keeping any liquid transfers simple (and manageable using a multichannel pipette).

A 96-well plate on ice

After electrophoresis and visualisation of the Schistidium ITS PCR products under blue, or U.V., light, the next step is to clean up these PCR products. This is where we remove unincorporated primers and dNTPs from the reactions. In our PCRs, we added all the ingredients in excess, so that there was more of everything than the reaction needed. This might seem wasteful, but compared to the costs of the time and plastics that would be required to carefully optimise each reaction, throwing lots in and getting quicker results really isn’t too profligate.

When a PCR is successful, the primer (an oligonucleotide, or short single stranded DNA molecule) binds to the end of the region that is getting copied, and a new strand of DNA is synthesised from the end of the primer, incorporating the primer into the new strand. Thus, primers get used up. The building blocks for DNA synthesis, the nucleotides (As, Ts, Gs and Cs), are the dNPTs (deoxynucleoside triphosphates) that we added to the PCR reaction, again in excess. So even when we’ve built a lot of copies, we still have some primers and dNTPS that weren’t used up.

This didn’t matter when it came to looking at the results on the gel, but does become important in our next reaction, the sequencing PCR. In the sequencing PCR, we add just a single primer, and we add a very precise blend of nucleotides. So we don’t want to carry over primers and dNPTs from the previous reaction. There are several ways of removing these.

The cheapest is through ethanol precipitation of the synthesised DNA, where the unincorporated primers and dNTPs stay in solution and are thrown away. This is less easy to scale up to plates, and the moment where you turn it all upside down and toss the liquid out is rather worrying – the cost of plates of subsequent sequencing failure if the DNA pellets were lost is huge, and the pellets tend not to be visible.

For several years we used a column-based approach, a bit like our DNA extraction method, where the PCR product is bound to a membrane, and the unincorporated primers and dNTPs are flushed off it, before the PCR product is eluted back into solution.

However, the method we now use most regularly involves a combination of two enzymes, Eco1, and Shrimp Alkaline Phosphatase (SAP), which work elegantly in combination, and which involves methodology that is easily scalable for working with plates and multichannel pipettes. The Eco1 enzyme digests single-stranded DNA, so it cuts the primers up into individual nucleotides. The SAP enzyme dephosphorylates the nucleotides (the dNTPs) so that they are no longer functional. Thus after using these enzymes, although nothing has been physically removed from the tubes, the unwanted reagents have been rendered unusable. For convenience, we buy in a commercial combination of the two enzymes called ExoSAP-IT™, and add a small amount of that to our reactions (in this case, around 1 μl ExoSAP-IT to about 15 μl of PCR product).

The enzymes work best at 37ºC, and so the reactions were put on a heating block for 15 minutes. The enzymes are killed at a higher temperature, and so the next step was to heat the reactions to 80ºC for 15 minutes, to make sure that no viable enzymes are left to interfere with the sequencing reaction. After that, the plate of cleaned Schistidium ITS2 amplicons was left in the fridge until it was needed for the sequencing reactions.

 

Links to reports on Moss diversity in an artificial landscape, an EU Synthesys Access project with Dr Wolfgang Hofbauer at RBGE:

Apr 112017
 

 

Gel loading in Lab 33

Once the polymerase chain reaction is over, it’s time to Run The Gel; this is make-or-break time, when we find out if our PCR amplification has actually worked.

The first step is to prepare and pour the gel. The gel is a 1-2% mix of agarose in a salt-containing buffer solution (for us, this is usually 1x TBE); the agarose is dissolved into the buffer using heat, in a microwave, and a bit of stain is added. We use about 5 μl of SYBRsafe for a 100 ml gel. The SYBRsafe binds to DNA and fluoresces under blue light, so we can see if we have produced amplified DNA.

After the agarose gel has cooled down to a comfortable temperature (so we don’t risk warping our ridiculously expensive plastic gel set-up with it!) we pour it onto a plate, and add a “comb” which sits in the gel and around which it will set, forming lines of small oblong holes in the gel’s upper surface into which we will later pipette our PCR product.

Setting up the loading buffer and PCR product in Lab 31

While waiting for the gel to set, we mix a small amount of the Schistidium ITS PCR product (3-5 μl) with a little loading dye (1-2 μl). The loading dye serves two purposes: it contains glycerol, which is rather dense and helps the liquid drop into the holes (wells) in our gel, and it also contains a colour (in this case, bromophenol blue), which means that we can see what we’re doing – dropping a colourless liquid through another colourless liquid into a colourless hole would otherwise be rather more an act of faith!

Using a manual multichannel to load the PCR product/loading solution

When the gel has set hard, we lift off the comb, taking care not to rip the gel (which is rather more brittle than gelatine-based jellies), and put the gel into a gel tank, covered with a thin layer of the TBE buffer that was used to make the gel. Then it’s time to load. Down one side, we put 3.5 μl of a “ladder”, a bought-in product containing DNA fragments of known lengths and quantities, which will act as a standard for the gel and let us estimate the sizes of any PCR products that show up. Adding the ladder, in which we know there is DNA, is also like a “positive control” that we’ve made our gel properly – if nothing at all shows up in the image of the gel, then it’s not proof that our PCR reaction failed, but that there was something wrong with our gel.

With the gel set-up that we are using for our plate of 96 Schistidium amplifications, we can load 8 samples at a time using one of the multichannel pipettes. For this, instead of our usual electric multichannels, we use a manual one so that we have more control over the rate of dispensation, and so we can stop if some of the liquid is not going in the right place: it’s quite a precarious operation!

Running our gel

Because the sugar-phosphate exerior of a DNA molecule has a negative electric charge, DNA molecules migrate through an electric field towards the positive electrode, and so if we apply a current to the gel, we can get the DNA to move through it. The filtering effect of the set agarose in the gel means that the DNA molecules migrate at a rate proportional to their size, with short molecules moving quickly, and longer ones more slowly. Generally, 30-45 minutes at 80 volts is enough to see if our PCR is successful.

After electophoresis, the gel is placed on the transilluminator

After the gel electrophoresis, the gel is carefully lifted out of the buffer, off the gel tank, and placed on a plate in a light box. (At this point with larger thinner gels like the one we ran for the Schistidium PCR products, it’s quite easy to break the gel.) Because the shape of the gel and of the light box are different, these gels need to be cut in two. Luckily, the halves fit nicely side by side in a single image.

Our gel beneath the filter

An orange filter is added, and when it’s turned on, the transilluminator shines with blue light that will make any DNA stained with SYBRsafe fluorescent, at which point a digital image is taken of the gel.

 

The first image of our Schistidium PCR products

To see what’s on the gel, we use an automated setting, where the computer choses the best exposure for the image. However, a bit of subsequent cropping and fiddling with the image itself can make for a far better gel picture. And the really good news for us is that, looking at our gel, it is clear that most of our Schistidium DNA extractions were successfully amplified for the ITS2 region in our PCR reaction!

The wells can be seen at the top of each row, as darker shadows; for the purposes of this image, the DNA has migrated towards the bottom of the screen. For only about seven of the 96 reactions is there no visible band of PCR product on the gel, and in the vast majority of cases a clear bright single band is present. (The fuzzier band that is sometimes visible a little further down than the ITS product, especially for the second last sample on this gel, is primer-dimer, an artifact caused during PCR, and not the amplified product we want; it’s far far shorter than the region we’re working with, as can be seen by comparing it with the size of our 1 kilobase DNA ladder).

Cropped and adjusted gel image picture, with a ladder on the right hand side of every third row

Links to reports on Moss diversity in an artificial landscape, an EU Synthesys Access project with Dr Wolfgang Hofbauer at RBGE:

Jul 192016
 
Decaying wooden fence, between concrete poles, Kufstein, Austria

Decaying wooden fence, between concrete poles, Kufstein, Austria

Recently in Kufstein, the home of Austrian bryologist Wolfgang Hofbauer, the demolition of an attractive old building and clearing of trees and other plants from the land, leaving a bare gravel patch used as a parking space, did have one interesting outcome: The new clearing led Wolfgang’s eye to a decaying wooden fence between concrete posts. Both the posts and the fence are partly covered in bryophytes, but among them, Wolfgang was very surprised to find the moss Schistidium growing on the old wood as well as on the concrete.

Schistidium on fence post, Austria

Schistidium on fence post, AustriaIn the bryological literature, the only reference to the plant growing on wood is a rare occurrence of Schistidium apocarpum, on lime-impregnated tree bark. The situation in this Kufstein parking lot seems unique, with at least two different species of Schistidium on the wood (although species identification is ongoing). Other more typical residents of old wood, which are also present, include Leucodon sciuroides, Orthotrichum affine and Hypnum cupressiforme. However, the unique assemblage is unlikely to last, as the climatic regime at the place will have changed following the removal of the trees, and the newly exposed rotten fence will probably soon be replaced.

Schistidium on fence post, Austria

Schistidium on fence post, Austria

Meanwhile, however, we wonder if similar unlikely assemblages of mosses are being observed elsewhere, and if there is an explanation for any potential changes in habitat?

 

 

Botanics Story and images provided by Wolfgang Hofbauer

 

Related literature

Wolfgang Karl Hofbauer, Laura Lowe Forrest, Peter M. Hollingsworth, Michelle L. Hart. 2016. Preliminary insights from DNA barcoding into the diversity of mosses colonising modern building surfaces. Bryophyte Diversity and Evolution 38(1).

Sam Bosanquet. 2010. Schistidium species reports, in: Atherton, Bosanquet & Lawley, Mosses and Liverworts of Britain and Ireland a field guide, British Bryological Society.

In plain sight – the mosses that grow on British walls. http://stories.rbge.org.uk/archives/19957

Hidden diversity in unexpected places – moss growth on modern building surfaces. http://stories.rbge.org.uk/archives/17489

 

Oct 082015
 

Back in 2014, staff in the molecular lab and herbarium at RBGE greatly enjoyed a three-week visit from Austrian Dr Wolfgang Hofbauer. With funding from the EU SYNTHESYS programme, Wolfgang, employed by the Fraunhofer Institute for Building Physics, was here to investigate mosses of modern building surfaces. He brought with him interesting chocolate, a taste for weird sodas, his wife and their son. While his family enjoyed the ambience of Edinburgh, strolling in the city and garden, Wolfgang was hard at work sampling mosses that he had collected in Austria, Germany and the UK, and comparing them to collections in our herbarium.

Schistidium, photographed by Wolfgang Hofbauer

Schistidium, photographed by Wolfgang Hofbauer

Building walls are a bit like cliffs, harsh habitats where only the very hardy can survive, with little shelter, periods of intense light and restricted water availability. Plants that do manage to establish there are frequently stunted individuals, with atypical morphology, which do not always possess the full range of characters (e.g. bryophyte sporophytes and spores) that may be required to identify species reliably. The group of mosses that Wolfgang is particularly interested in, Schistidium, grow slowly on buildings, with frequently incomplete development. In the past, collections of these mosses have mostly been named as Schistidium apocarpum, a species that is thought to be widespread and common. Wolfgang’s work involves comparing well-developed samples of Schistidium species from our herbarium with the stunted individuals he has been collecting from walls, cement and tarmac, both by looking at the plants’ morphological features, and by comparing standard regions of their DNA (plant DNA barcoding). An important part of this work is the development of a “reference” set of sequences from fresh and herbarium samples that possess a full range of morphological characters, allowing them to be identified reliably to species. Many of the Schistidium collections in Edinburgh have been revised by one of the most respected taxonomists working on the group, Dr Hans Blom, allowing us to generate DNA sequences from some of the plants that Blom has studied and named; these should correspond to Blom’s taxonomic ideas about species in Schistidium. This standard reference set will enable future identification of stunted and dwarfed specimens from building substrates, by comparing their DNA sequences to the standards.

Modern building surface with bryophyte growth, photographed by Wolfgang Hofbauer

Modern building surface with bryophyte growth, photographed by Wolfgang Hofbauer

From the work we conducted with Wolfgang during his visit to RBGE, we could see that the first Schistidium colonisers on modern building surfaces actually include several distinct genetic lineages. These lineages have been sampled from several locations around Europe, but in order to define geographic patterns and to evaluate morphological characters, we need more intense sampling, which will allow us to discuss the diversity and ecology of Schistidium colonisers on modern building surfaces. There are two practical outcomes of isolating the lineages that occur on these surfaces. First, if we can identify dominant species, then we may be able to identify selective biocontrol of moss growth on building surfaces, which can be regarded as unsightly. Second, and in contrast, some of these lineages may actually be adapted to grow on modern building surfaces – and therefore could be used for moss gardening. This could be beneficial for insulation purposes, to reduce air pollution and climate gases (CO2 scavenging), to increase biodiversity within an area, and, last but not least, for aesthetic purposes: greening cities and visually breaking up expanses of concrete.

(This Botanics Story has been cowritten by Laura Forrest and Wolfgang Hofbauer.)

 

Wolfgang Karl Hofbauer, Laura Lowe Forrest, Peter M. Hollingsworth, Michelle L. Hart. 2016. Preliminary insights from DNA barcoding into the diversity of mosses colonising modern building surfaces. Bryophyte Diversity and Evolution 38(1)