High altitude PCR 2: preliminary data!

As promised on Friday, here is some initial data for this idea.  I still don't know if it truly is an effect of altitude or just denature temperature, but here is what I did.  I set up 8 identical reactions (10uL each) to amplify an 800bp product from pUC19 and ran them under three different cycling conditions on the same cycler on the same day (barometer = 30.43 in Hg, corrected boiling point = 93.38C).  I note that there was a lot of condensation present on the upper portion of the wells following Cycle 1, but much less after Cycle 2 or Cycle 3.

Cycle 1
1) 95C 1 min
2) 95C 30s
3) 55C 30s
4) 72C 45s
5) goto 2, 29X

Cycle 2
1) 90C 1 min
2) 90C 30s
3) 55C 30s
4) 72C 45s
5) goto 2, 29X

Cycle 3
1) 90C 1 min
2) 80C 30s
3) 90C 30s
4) 55C 30s
5) 72C 45s
6) goto 2, 29X

I ran 2uL each product on a gel:

Everything looks about the same, but you might notice there are more large (greater than 800bp) non-specific products in the 95C denature and the 80C/90C denature programs than the 90C denature alone.  I think this is partially because 30 cycles may have been too many for this amplicon.  I hope to rerun this analysis with fewer cycles another day.

That said, significance was achieved (ANOVA p = 0.0104)!  Again, I think this is largely due to the added non-specific products.  Here is a nice bar graph showing the results with Tukey's HSD separations denoted. The concentrations were obtained using OD260 method following ethanol precipitation of all products.  Each sample was resuspended in 50uL 1X TE.

What can I take away right now?  90C is adequate for denature of a simple bit of DNA such as pUC19 and I know it avoids boiling my sample at this altitude.  Less condensation appears on the upper portion of the tube walls under a 90C denature, presumably preventing concentration of reagents which can lead to production of non-specific bands.  The effect, regardless of its cause, results in a significant difference in total PCR yield, but presumably more of the product in the 90C denature is the product I want while the other cycles produce unwanted by-products.

Where to go next?  Repeat with fewer cycles, possibly use a qPCR to guide the appropriate number of cycles, measure with a pipet the volume of reaction that remains at the bottom of the well following each cycle next time.  For now, reduce denature step to 90C.  When this particular reaction works a bit better, test it again at different altitudes.  Also maybe test a more complex target.

At the very top of Snowbowl (~11,500ft, if they will loan me an outlet for the day), the boiling point would be 89.2C on the same day, thus boiling all of my reactions.  In the Verde valley (~3,500ft), the boiling point would be 96.9C thus eliminating all boiling for all reactions.

A colleague told me this weekend his new motto is "free Tibet (to do PCR)!"

High altitude PCR

It occurred to me the other day that some unreliability among PCR samples that we experience here in Flagstaff may be attributable to the altitude here (7000ft). I did some googling on high altitude PCR but came up with pretty much nothing. Our lab has 4 Biorad iCyclers, 1 Biorad iCycler for qPCR, 1 BioRad Tetrad (MJ style with 4 blocks), 1 MJ Research PTC-200, and 2 MJ Research PTC-100s.

I first dug into the manuals to try to find anything about altitude correction factors. Nothing. The closest I came was finding that they could be "safely used" at altitudes up to 2000m (~6500ft). That gets almost all the way up to Flagstaff, but says nothing about whether there are any altitude-related issues I should be thinking about.

I then called BioRad tech support and chatted with a rep for a bit on this issue. She was a little amused and quickly regurgitated the ideal gas law (PV=nRT) which would have taken me a little longer to come up with. She pointed out that there are temperature sensors in all of the blocks of the machines we use, so the temperature reported by the cycler is correct as long as you accurately enter the volume of your reaction so it can correctly calculate the sample temperature from the block temperature probes.

I then pointed out that water boils at just under 93C here in Flagstaff (assuming barometric pressure of 23 in. Hg). If we are experiencing high atmospheric pressure (most of the time), the boiling point is slightly higher (closer to 95C) or if we have a storm (low atmospheric pressure) it could go as low as 91C. What this means is that every person here at NAU who follows the prescribed protocol for their PCR is boiling their reactions at every denature step. Since the denature temperature for most programs is still 94-95C, this is just the cusp of the boiling point on most days (it is usually sunny here). It also is not above the magic point at which you will significantly degrade the half-life of your enzyme with each step (about 94-95C). The boiling can still have a significant effect on the reaction by changing the concentration of your buffer (increased concentration of all reagents). Sometimes the moisture is retained due to the heated lid preventing condensation on the upper part of the tube and the lid compression preventing any gaseous escape, but more often than not, your reaction will contain less fluid at the end of the cycle than you began with.

Example: If you perform 10uL reactions and after 35 cycles you have 8uL, your MgCl2 concentration (to pick a reagent) will increase from 2mM to 2.5mM over the course of your cycle. Excess of MgCl2 can contribute to mis-priming and production of non-specific products. Excess KCl can produce unwanted short non-specific products. Excess polymerase can result in all kinds of background (a smeared appearance). Some environmental DNA samples such as are often processed in our lab also contain a lot of PCR inhibitors (e.g. polyphenols). The evaporation can therefore increase the endogenous inhibitor concentration to a point that effectively stops the reaction somewhere along the cycle.

You get the idea. Evaporation BAD!!!(say it like Dana Carvey playing George HW Bush)  Though she couldn't provide me a concrete solution to this concern short of an artificially pressurized laboratory, she offered two suggestions that sounded very good to me:

1) Persons performing PCR at high altitude should reduce their temperature of denaturation according to the local atmospheric pressure. For our altitude, she suggested 90C. I asked if this time should be extended slightly to account for the lower temperature and she said she did not know, but it probably wouldn't hurt...which takes us to her second suggestion.

2) PCR (generally speaking at all altitudes) of GC-rich regions should include a "pre-denature" step of 80C for 1 min to "slow" the denature step to allow for the disentanglement of complex secondary structures and eventual denaturation of the GC-rich region. I should point out that she offered this as a complete alternative to ever adding DMSO to your reaction which, as you may know destabilizes hydrogen bonding, thus allowing efficient denaturation of GC-rich regions, but then also complicates your annealing step. She claimed the 80C for 1 min will solve this problem without messing with your annealing and thus may also be useful for high-altitude PCR.

What I learned: At 7000ft, reduce the annealing temperature to 90C. I can probably keep my denature time at 20-30 sec, but a pre-denature step of 15-30 sec at 80C may improve overall reaction efficiency with no further changes (longer if PCR target region is GC-rich).

Happy high altitude PCRing!!