PROTOCOLS FOR SOME USEFUL
PCR-BASED MOLECULAR SYSTEMATIC APPROACHES

Harvey E. Ballard, Jr.
Department of Environmental and Plant Biology
Porter Hall, Ohio University
Athens, OH 45701 USA

Below are links to detailed protocols for several widely used, or useful, molecular systematic approaches to address organismal questions. All are PCR-based--e.g., full-scale restriction site analysis using Southern blotting and labeled probes is not described here. I have restricted protocols to PCR-based methods, including some which utilize an automated sequencer for greatest efficiency, for a number of reasons. First, they require little or no prior laboratory experience and are therefore ideal to teach both undergraduate and graduate students. Second, those not relying on a sequencer are comparatively cheap to apply. Third, those noted in the above reason also require minimal laboratory setup and cost, making them especially appropriate for students who will ultimately join labs with limited available resources. We particularly use DNA sequencing and ISSRs extensively, and we are also developing AFLPs for use in a variety of projects.

Protocols are provided in Word for Windows '97 format; associated data sheets and other tables are in Word or Excel format, below the protocols. Details relate specifically to our laboratory facilities, and the lab supplies and consumables we presently use, but other protocols or kits can be substituted with no problem. In particular, any means of extracting genomic DNA should work, as long as the extract is reasonably clean; AFLPs, however, will require very clean extracts, which may require you to do two chloroform-isoamyl alcohol extractions or use a spin-column. Similarly, a wide variety of cleaning procedures are available for PCR product cleanup in preparation for cycle-sequencing, if you are doing DNA sequencing, and these are more or less equally effective at removing primers and other PCR constituents. Quantification of DNA is helpful or even essential; for AFLPs it may be best to use a fluorimeter or spectrophotometer to obtain the greatest accuracy, but for cycle-sequencing or comparison of ISSR samples, for instance, electrophoresis in an agarose gel with a mass ladder standard may provide sufficient accuracy for your needs. The protocols linked to the table below will describe the particular methods we have adopted--especially the easiest and cheapest methods which work adequately for our purposes and are scientifically justifiable.

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Links to Protocols
1. Alternative DNA extraction protocol--We use a Promega "Wizard" kit to extract genomic DNA, but this protocol works reasonably well, at least for PCR amplification prior to sequencing; it may not be adequate for preparing extracts to be used for ISSRs or AFLPs (should be tested with few samples first)

2. Inter-Simple Sequence Repeats (ISSRs)--nuclear "anonymous" fragments generated by a PCR reaction using a single primer, which has a microsatellite (=tandem repeat) embedded within it; appropriate for population-level studies among very closely related individuals, or to detect species-specific alleles in hybridization or potential introgression; simple and relatively inexpensive, relying only on a PCR reaction (for 3-4 primers per individual) and agarose gel electrophoresis to visualize fragments; dominant markers amenable to distance methods of analysis and modified F-statistics ("phi"-statistics)

3. Amplified Fragment Length Polymorphisms (AFLPs)--fragments generated by a combination of restriction site digestion and successively selective amplifications with increasingly long primers; appropriate for studies of population or closely related species, or to detect species-specific alleles in hybridization or potential introgression; relatively long series of steps [much pipetting!], and expensive both because of several costly constituents and because of reliance on an automated sequencer; more or less dominant markers amenable to distance methods of analysis and modified F-statistics ("phi"-statistics)

4. PCR-based Restriction Fragment Length Polymorphisms (PCR-RFLPs)--nuclear or plastid fragments, depending on gene regions selected, generated by PCR-amplification of known gene regions followed by restriction enzyme digestion to access restriction site variation; appropriate for phylogenetic studies of related species or perhaps closely related genera; relatively inexpensive, relying on PCR reactions and restriction enzymes; amenable to discrete ("cladistic") approaches

5. DNA Sequencing--nucleotide (and possibly insertion/deletion) variation generated by PCR-amplification and cycle-sequencing of a known gene region, then analysis on an automated sequencer; appropriate for species- and higher-level phylogenetic studies, occasionally among geographically separated populations for older groups; somewhat expensive because of analysis on an automated sequencer; amenable to discrete ("cladistic"), probabilistic and other approaches

6. Other Useful Documents

For lectures on various aspects of molecular systematics and highlights of many studies utilizing various types of comparative molecular data, visit my web site for the "Molecular Approaches to Ecology, Systematics and Evolution" course, PBIO 469/569.

Prepared by H. Ballard; last revised 8 February, 2001

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