Procedures: Microsatellite-primed PCR
The interrepeat-PCR strategy combines elements of Alu-PCR, microsatellite and RAPD (Meyer et al. 1991, Meyer et al. 1993). In this approach oligonucleotides complementary to microsatellites, that is, tandem repeats of short sequence motifs (mainly 2-4 nucleotides), serve as single primers. If inversely repeated microsatellites are located within an amplifiable distance of one another, the inter-repeat sequences are amplified. This technique, so-called microsatellite-primed PCR or interrepeat PCR, has been used successfully to amplify hypervariable repetitive DNA sequences in a wide range of animal, plant, and fungal species (Meyer et al. 1993; Meyer and Mitchell 1995; Weising et al. 1995). In this page, we describe a protocol for amplifying intermicrosatellite DNA regions of ectomycorrhizal fungi (see also Martin et al. 1996). These PCR fingerprintings allowed distinction among different fungal taxa at an interspecific as well as intraspecific level.
Microsatellite-primed PCR presents several advantages of RAPD analysis (e.g. no need for sequence information) and of microsatellite analysis (e.g. use of high-stringency annealing conditions leading to reproducible DNA patterns), although this latter contention has recently been challenged (Weising et al. 1995).
A single microsatellite primers (e.g. (CA)8, (CT)8, (GT)8, (GAC)5, (GTG)5, (GACA)5 or (TGTC)5) is usually used to direct the amplification of inter-repeat regions. However, the primer could be used in conjunction with another microsatellite primer.
- Dilute the DNA solution with sterile de-ionized water. Usually, three 10-fold dilutions are tested for each sample, 1/10, 1/100, and 1/1000.
- Prepare enough PCR master mix for the number of PCR assays to be performed, plus 10% to take in account pipeting inaccuracies. Assays are usually performed in total volume of 33 ul (30 ul master mix + 3 ul template DNA). For 30 ul of master mix, add 3 ul of 10X PCR Appligene buffer, 1 ul of 10 mM dNTPs (200 uM final concentration), 1 ul of 100 uM of microsatellite primer, and 0.2 ul of Taq DNA polymerase (5 U/ul). Mix well and distribute in the required number of assay tubes.
- Add 3 ul of DNA crude extracts to each assay tube. If needed, overlay with a drop of light mineral oil (Sigma #M5904). Cap each tube carefully and immediately label each tube, on the cap, with a water-resistant marker. Write down the description of each assay with its corresponding label.
- Place in the thermal cycler and start the PCR. The cycler should be programmed to perform a initial denaturation step at 94°C for 3 min, and then 8 cycles at 94°C for 0.5 min (denaturing step), `touchdown' annealing temperature for 0.5 min, and 72°C for 1 min (extension step). For example, the thermal cycling starts at an annealing temperature of 65°C and is reduced by 1°C at each subsequent cycle to 57°C. This is followed by an additional 25 cycles at 94°C for 0.5 min, 58°C for 0.5 min, and 72°C for 1 min, and then hold at 72°C for 10 min. Assay tubes should be kept at 4°C until analyzed.
- When the optimal annealing temperature is determined, regular PCR cycling could be used: 94°C for 3 min, and then 30 cycles at 94°C for 0.5 min, 55°C (or any optimal temperature between 50 and 65°C) for 0.5 min, 72°C for 2 min, and then hold at 72°C for 10 min.
- Prepare either agarose (1.5% NuSieve/0.5% Molecular Biology grade agarose in TBE buffer) or 15 % polyacrylamide gels according to Sambrook et al. (1989) to analyze the amplicons.
- Spot many 1 ul droplets of gel loading buffer on a piece of Parafilm M (Prolabo #01.963.003). Carefully pipet 7 ul of amplified mixture through the oil (if any), mix with a droplet of loading buffer by pipeting in and out on the Parafilm, and load in a well of the gel. Repeat for all samples, including a size standard . Size standards are loaded in the first and last well. Note the content of each well, run the electrophoresis until the Bromophenol blue migrates 2/3 of the gel.
- Immerse the gel in an ethidium bromide staining solution (4 ug/ml) for 15-30 min, rinse in water for 15-30 min, visualize on a UV transilluminator and photographed using a Polaroid camera and 667 film or a video computerized system.
- Grube M, Depriest PT, Gargas A, Hafellner J (1995) DNA isolation from lichen ascomata. Mycol. Res. 99: 1321-1324
- Heath DD, Iwama GK, Devlin RH (1993) PCR primed with VNTR core sequences yields species specific patterns and hypervariable probes. Nucl Ac Res 21: 5782-5785
- Martin F, Costa G, Delaruelle C, Diez J (1996) Genomic Fingerprinting of Ectomycorrhizal Fungi by Microsatellite-primed PCR. In `Mycorrhiza manual' (A Varma, B Hock, edts), Springer Lab Manual, in press
- Meyer W, Koch A, Niemann C, Beyermann B, Epplen JT, Börner T (1991) Differentiation of species and strains among filamentous fungi by DNA fingerprinting. Current Genetics 19: 239-242
- Meyer W, Mitchell TG, Freedman EZ, Vilgalys R (1993) Hybridization probes for conventional DNA fingerprinting used as single primers in the polymerase chain reaction to distinguish strains of Cryptococcus neoformans. J Clin Microbiol 31: 2274-2280
- Meyer W, Mitchell TG (1995) Polymerase chain reaction fingerprinting in fungi using single primers specific to minisatellites and simple repetitive DNA sequences: strain variation in Cryptococcus neoformans. Electrophoresis 16: 1648-1656
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning; A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, New York
- Weising K, Atkinson RG, Gardner RC (1995) Genomic fingerprinting by microsatellite-primed PCR: a critical evaluation. PCR Meth Applications 4: 249-255