Polymerase Chain Reaction

VGEC: Student Notes PCR Tutorial – use of graphs as learning objects Dr Celia A May, Department of Genetics, University of Leicester

You have been given a handout by your tutor, and based on that and the information you have just seen in the brief video, attempt to answer the questions below. It is best to do this in small groups of 2 or 3 per group.

Exercise 1: Basic PCR a) A PCR is performed using a primer pair that selectively amplifies a 100bp fragment from human total genomic DNA.

 What shape graph would you expect if you plotted target copy number against cycle number? Sketch your predicted graph.  What factors influence the shape at very high cycle numbers, e.g. 40+ cycles? b) A second PCR is performed now using a primer pair that selectively amplifies a 10kb fragment from total genomic DNA.

 How would this graph compare to the first? Add this plot to your first graph.  Can you think of factors that might give rise to differences in the two graphs?

Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/ Exercise 2: Quantitative PCR

PCR can also be performed on cDNA prepared from specific cell sources, allowing gene expression levels to be evaluated. Such PCR quantification, “qPCR”, is usually carried out in “real-time”, that is the increase in DNA can actually be monitored as it is amplified. This can be achieved by a number of means, the simplest of which is to include a fluorescent dye in the reaction, which will specifically bind double-stranded DNA and emit light when excited by a laser beam. The amount of light emitted should be proportional to the amount of double-stranded product present at any given cycle and would be monitored at the end of the extension step

Why do you think we monitor at this step? a) Two real-time PCR reactions are performed using a primer pair that selectively amplifies a 60bp fragment of a given tissue-specific gene. The first reaction is seeded with 100ng of cDNA prepared from cell type X and the second is seeded with 100ng of cDNA from cell type Y.

 Plot the graphs you would expect to see for these two reactions on the same axes. Account for any differences in the two plots. b) A series of real-time PCR reactions are now conducted using the same primer pair but with just the cDNA derived from cell type X. A two-fold serial dilution is performed such that reactions are either seeded with 100ng, 50ng, 25ng or 12.5ng of this cDNA.

 Sketch the graphs you would expect to see for these four different PCR reactions on the same set of axes.

 Over which cycle range would you expect to see a quantitative response?

c) The reaction seeded with 50ng of the cDNA gives a fluorescence signal of 106 units at 20 cycles.

 At what cycle number would you expect each of the other reactions to reach this same threshold of fluorescence?

d) cDNA samples from X cells have been isolated from individuals A and B. Each of these samples is used to seed two different qPCR reactions. The

Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/ first specifically amplifies the 60bp fragment described above. The second specifically amplifies a 58bp fragment of a housekeeping gene.

 If the PCR from individual A reaches a given threshold of fluorescence 4 cycles before that of individual B for the 60bp product, yet just one cycle earlier for the 58bp product, what conclusions would you make about the relative expression of tissue-specific gene in these two individuals?

What assumption are you making about the kinetics of the housekeeping gene PCR reaction?

Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/