Separation of the 8 major molecular types of the
C. neoformans complex via PCR-fingerprinting
W. Meyer
Materials
· Genomic DNA (10 ng/ml)
· 10x PCR Buffer: 10 mM Tris-HCl pH 8.3, 50 mM potassium chloride, 1.5 mM magnesium chloride (Perkin Elmer, Applied Biosystems Division, Foster City, CA, USA)
· Deoxynucleotides (dNTPs): 2mM each of dATP, dTTP, dCTP, dGTP (Boehringer, Mannheim, Germany)
· 50 mM magnesium acetate (Sigma Chemical Co. St. Louis, MO, USA)
· Primer (10 ng/ml): either M13, (GTG)5 or (GACA)4
· AmpliTaq DNA polymerase (5 U/ml) (Perkin Elmer, Applied Biosystems Division, Foster City, CA, USA)
· Sterile deionised water
Methods
PCR-fingerprinting is performed with three different primers in order to ensure the results obtained with each are reproducible. M13 is a minisatellite specific primer derived from the core sequence of the wild-type phage M13 (5' GAGGGTG GCGGTTCT 3'), while (GTG)5 and (GACA)4 are microsatellite specific oligonucleotides; those sequences can be used as single primers in PCR-fingerprinting of C. neoformans (Meyer et al, 1993).
Each PCR reaction is performed in a 0.5 ml thin walled reaction tube in a 50ml volume containing:
31.0 ml sterile deionised water
2.5 ml of DNA (10 ng/ml)
16.5 ml of master mix:
· 5 ml of 10x PCR buffer
· 5 ml of dNTPs
· 3 ml of magnesium acetate (50 mM)
· 3 ml of primer (10 ng/ml)
· 0.5 ml AmpliTaq
1 drop of light white mineral oil
The PCR reactions takes place in a Perkin Elmer model 480 thermal cycler:
35 cycles Denaturation 94°C for 20 seconds
Annealing 50°C for 1 minute
Extension 72°C for 20 seconds
1 cycle Final extension 72°C for 6 minutes
Soaking cycles 5°C for
The PCR products are visualised on a 1.4 % agarose electrophoresis gelat 60V for 14 cm.
Notes:
· Optimisation of the PCR-fingerprinting procedure (Meyer et al, 1993) has found empirically that supplementing the PCR reactions with 3 mM magnesium acetate increases the reproducibility of fingerprints obtained by amplification of fungal DNA.
· All DNA and reagents were kept on ice during the preparation of reactions to reduce the risk of DNA degradation, enzyme inactivity or non-specific amplification.
· In order to minimise the risks of DNA contamination, all reactions were prepared in a specialised DNA workstation (UniEquip, Munich, Germany), using designated pipettes and tips. The DNA workstation is fitted with a filter system for removal of DNA aerosols and a UV light for pre- and post-work decontamination. A negative control was performed with the PCR reactions, containing water in place of DNA. When the PCR products were separated on a gel, an empty lane for the negative control was indicative of no contamination.
· Care was taken to ensure the same thermal cycler was used for all PCR procedures. Different brands and models of thermal cycler may affect the amplification of DNA due to differences in the heating blocks.
Figure: PCR-fingerprints generated with the primer M13 obtained from the reference strains of the major C. neoformans molecular types.
Reference:
1) Meyer W & Mitchell TG (1995): PCR fingerprinting in fungi using single primers specific to minisatellites and simple repetitive DNA sequences: strain variation in Cryptococcus neoformans. Electrophoresis 16:1648-1656
2) Meyer W, Marszewska K, Amirmostofian M, Igreja RP, Hardtke C, Methling K, Viviani MA, Chindamporn A, Sukroongreung S, John, MA, Ellis DH & Sorrell TC (1999): Molecular typing of global isolates of Cryptococcus neoformans var. neoformans by PCR- Fingerprinting and RAPD - A pilot study to standartize techniques on which to base a detailed epidemiological survay. Electrophoresis 20 (8): 1790-1799.
3) Ellis D, Marriott D, Hajjeh RA, Warnock D, Meyer W & Barton R (2000): Epidemiology: surveillance of fungal infections. Med. Mycol. 38 (Supp. 1): 173-182.
4) Meyer W, Castaneda A, Jackson S, Huynh M, Castaneda E and the IberoAmerican Cryptococcal Study Group (2003): Molecular typing of IberoAmerican Cryptococcus neoformans Isolates, Emerging Infect. Dis.: 9(2):189-195. 44)