20.109(S13):PCR and paper discussion (Day3)

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Revision as of 14:06, 10 January 2013

20.109(S13): Laboratory Fundamentals of Biological Engineering

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DNA Engineering        Protein Engineering        Cell Engineering              



In order to select and amplify short DNA fragments from individual bacteria (or later, microsporidia), you will use the polymerase chain reaction, PCR. PCR comprises three main steps: 1) template DNA containing a desired sequence is melted, 2) primers anneal to specific locations on the now melted (i.e., single-stranded) DNA, and 3) the primers are extended by a polymerase to select and create the desired product. Extension occurs at ~70 °C, melting at ~95°C, and annealing at a temperature ~5 °C below the primer melting temperature; thus, the repetition of these steps is called thermal cycling. After each cycle, the newly formed products themselves become templates, causing exponential amplification of the selected sequence. (Note that the early rounds of PCR will not produce the desired product - we will see why in today's pre-lab lecture.)

Kary Mullis
Kary Mullis



all or some needs to go right on D1? hard to understand primers otherwise...

We can improve the reliability and accuracy of PCR, especially important when the DNA originates from a stool sample, by two key methods. The first is the use of a highly specific polymerase, one with either explicit or inherent hot start properties. Hot start means that the polymerase is inactive at low temperature (add some further elaboration here), while the reaction is being set up and initially heated, thus reducing non-specific interactions between primers and non-target DNA. The important point to note here is that the target DNA (DNA of interest) may be present at a low concentration compared to the DNA in the sample as a whole, increasing opportunities for non-specific binding. The second performance enhancer is adding bovine serum albumin (BSA) to the PCR. As you saw if you clicked on the Kreader paper linked on Day 2, BSA itself binds many inhibitors of PCR, thus acting as a competitor with respect to the polymerase. We would much rather that inhibitors bind the BSA than bind the polymerase and interfere with its function! BSA is hydrophobic and somewhat positively charged, making it a great non-specific binder of proteins that we will use time and again in 20.109.

Even taking the above precautions, you might expect to obtain some non-specific products in your PCR today. Next time, you will run your entire reaction mixtures through a gel to visualize them, and then excise and purify the band of the correct size (~1400 bp).


Scanning EM image of agarose polymer
Scanning EM image of agarose polymer


One parameter that affects the way DNA travels through a gel is the pore size, which is in turn affected by both the weight percent of the gel and the type of agarose used. Because we are separating large DNA fragments (> 1 Kbp) in the bacteria experiment, a low percentage (namely 1%) gel is appropriate. In the microsporidia experiment, small fragments (~ 0.1 Kbp) are expected and thus a high percentage (namely 3%) gel will be used. Specifically, we will use a high-resolution (HR) agarose; its low viscosity means that high weight percent solutions are tractable to work with, and that the solidified gel remains pliable rather than brittle. HR agarose can be prepared by chemically modifying and/or partially depolymerizing natural agarose (as described here).

You will melt the agarose gel bands, then isolate the DNA by using a silica (SiO2) column similar to the one you used last time. Salt concentration and pH effects, along with ethanol precipitation, will alternately allow for binding and eluting the DNA while washing away contaminants. The final elution here will be done in water, and these silica columns will collect any DNA between about 70 and 10,000 bp.save some of this for next time?

The PCR will run during the whole lab period. In the meantime, we will discuss a journal article, both to learn more about phylogenetic analysis and to become comfortable reading and discussing the primary scientific literature. In 2-3 weeks, you will each present an article on your own.


Part 1: Prepare PCR to detect bacterial 16S


  2. Pre-chill the tubes on a cold block.
PfuUltra buffer (10X stock) 5
Primer mix 1
dNTPs 1
10% [5%?] BSA (100X stock) 0.5
Water 32.5
DNA template 5 N/A
Segment Cycles Temperature (° C) Time
1 1 95 5 min
2-4 35 95 1 min
51 1 min
72 2 min
5 1 72 10 min
6 1 4 indefinite

Part 2: WAC Session


Part 3: Journal article discussion


Technical Background

Discussion Topics




Probably changing to assigning these in advance and doing slightly more formal presentation (single slide prepared)

When you arrive in lab today, each group will be assigned one of the following topics to present to and discuss with the rest of the class. You should be somewhat familiar with the whole WHOEVER paper by now, but will have some time in-class to refresh your memory and become the resident expert in one of the following areas.

For next time

Reagent list

  • PfuUltra polymerase and buffer from Agilent
  • Primers
    • F8-27 sequence: 5' AGAGTTTGATCCTGGCTCAG
    • R1392-1407 sequence: 5' ACGGGCGGTGTGTACA
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