Friday, 14 March 2014

First week of the Synthetic Biology Project: PCR success!

It was a foggy on Thursday morning when I walked into the Innovation labs and Michael was distributing the reagents, template, primers, dNTPs etc. The Y12 group were due to arrive in an hour and by the end of the afternoon, all of their skills training would be put to the test. I also had to fit in an explanation of the fundamentals of DNA replication: the enzymes, the concept of primers and the polarity of DNA synthesis, not forgetting the logic of the thermal cycling process. By 10am the student teams had assembled all of the reactions and the new PCR machines were cycling (only for the second time!). By 1.30pm the gels had run and the results were in! Out of the 16 groups we had two failures, which I think is pretty impressive and one of the gels is shown on the left. No need for labels, the PCR product is clear in all but one reaction mixture. This means that we have now worked through all but one of the Y12 target methods, (ligation and transformation the only experimental method that we have yet to complete before Easter vacation). With these methods in hand we can now isolate the gene encoding non-ribosomal protein synthesising machinery from our Blue-green algae. Moreover, Y13 is set to become a great opportunity to exploit these skills and pursue a range of customised projects.

But back to Thursday. The Polymerase Chain Reaction (PCR) results in the sequential amplification of DNA through the polymerisation of new DNA strands, driven by a heat stable (thermostable) DNA Polymerase. Two strands of the template duplex are denatured and form the templates for primers and polymerase to generate copies. Two strands become, 4, 4 become 8, 8 become 16 and so forth until millions of copies are produced in a matter of hours. Not only does this facilitate DNA analysis, it is the core of many diagnostics and forensic methods. In short, it is possibly the most widely used method in Life Sciences today. But it also provides an opportunity to appreciate mathematical relationships.

DNA amplifies in an exponential manner, but the use of logs and their value in plotting data that span large experimental ranges, is not covered by the National Curriculum, unless you take Maths A level. No surprises then that understanding pH, or estimating binding affinities and physical relationships in Biology, essential in many diagnostic and analytical procedures, proves so challenging for first year undergraduates  and new lab technicians. So I am delighted to say that we used our time in between amplification and electrophoresis, to explore the value of logarithmic plotting methods (a picture of school log tables is shown left, for those of you over 50!). We plotted fragment migration on gels, we have previously looked at bacterial growth curves and what was really encouraging was the enthusiasm the students expressed for "having a go" at something that is often perceived to be too challenging. So, not only did we get the technique of end point PCR nailed, we have paved the way for understanding real time PCR analysis and more generally, non-linear experimental data, which pervade Biology. 

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