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Template preparation for rapid PCR in Colletotrichum lindemuthianum

Preparação de DNA molde para PCR rápido em Colletotrichum lindemuthianum

Abstracts

Isolation of DNA for PCR is time-consuming and involves many reagents. The aim of this work was to optimise a rapid and easy PCR methodology without previous DNA isolation. Different strains of the phytopathogenic fungus Colletotrichum lindemuthianum were used. Protoplasts were generated using lytic enzymes under high incubation temperatures using different methodologies to obtain the template. A rapid (10 minute) methodology was successful for smaller amplicons (<750 bp).

anthracnose; hygromycin resistance; internal transcribed spacer; rapid screening; PCR methodology


A extração de DNA para realização de PCR é demorada e envolve vários reagentes. O objetivo deste trabalho foi desenvolver uma metodologia rápida e fácil que permita utilizar a técnica de PCR sem necessidade de extração de DNA. Diferentes isolados de Colletotrichum lindemuthianum foram utilizados. Para obtenção do DNA moldes foram testados protoplastos obtidos através do uso de enzimas líticas, incubados sob altas temperaturas e diferentes metodologias. Uma metodologia rápida (10 minutos) foi desenvolvida para produtos de PCR menores (<750 pb).

antracnose; espaços transcritos internamente; metodolologias de PCR; resistência a higromicina; triagens rápidas


Template preparation for rapid PCR in Colletotrichum lindemuthianum

Preparação de DNA molde para PCR rápido em Colletotrichum lindemuthianum

M. Gabriela Roca* * Corresponding author. Mailing Address: Departamento de Biologia. Universidade Federal de Lavras, Caixa Postal 37. 37200-000, Lavras, MG, Brasil. E-mail: lcdavide@ufla.br ; Lisete C. DavideI; Alan E. WhealsII

IDepartamento de Biologia, Universidade Federal de Lavras, Lavras, MG, Brasil

IIDepartment of Biology and Biochemistry, University of Bath, United Kingdom

SHORT COMMUNICATION

ABSTRACT

Isolation of DNA for PCR is time-consuming and involves many reagents. The aim of this work was to optimise a rapid and easy PCR methodology without previous DNA isolation. Different strains of the phytopathogenic fungus Colletotrichum lindemuthianum were used. Protoplasts were generated using lytic enzymes under high incubation temperatures using different methodologies to obtain the template. A rapid (10 minute) methodology was successful for smaller amplicons (<750 bp).

Key words: anthracnose, hygromycin resistance, internal transcribed spacer, rapid screening, PCR methodology

RESUMO

A extração de DNA para realização de PCR é demorada e envolve vários reagentes. O objetivo deste trabalho foi desenvolver uma metodologia rápida e fácil que permita utilizar a técnica de PCR sem necessidade de extração de DNA. Diferentes isolados de Colletotrichum lindemuthianum foram utilizados. Para obtenção do DNA moldes foram testados protoplastos obtidos através do uso de enzimas líticas, incubados sob altas temperaturas e diferentes metodologias. Uma metodologia rápida (10 minutos) foi desenvolvida para produtos de PCR menores (<750 pb).

Palavras-chaves: antracnose, espaços transcritos internamente, metodolologias de PCR, resistência a higromicina, triagens rápidas

The fungus Colletotrichum lindemuthianum (Sacc. & Magn.) Scribner is the causative agent of anthracnosis, one of the most important diseases of the common bean in Brazil (14). Due to its economic damage and widespread abundance, this fungus has been subjected to intensive study with the aim of developing both new varieties of beans that are genetically resistant to C. lindemuthianum as well as understanding the genetic basis of pathogenicity.

PCR (Polymerase Chain Reaction), that allows the characterization of the DNA of individual or populations, is among methodologies that have been applied to the study of genetic variability of fungi. The most time-consuming requirement of this process is usually DNA extraction and this can hinder its effective deployment when either a large number of samples need to be analysed or when there is only a small amount of material for DNA extraction. Some DNA extraction protocols for fungi use toxic chemicals (17), are time consuming while disrupting the cell or need excessive amounts of material (2); and sometimes it is difficult to release the cellular contents from mature hyphae or spores (19). Fungi with slow growth rates require growing cultures in liquid media in flasks or material that can be scraped from cellophane agar (17). Some protocols facilitate using new methodologies, with material such as 'reverse agar' (15), using standard DNA extraction protocols (8) or using fresh material (3). When spores are available suitable and quick methodologies have been developed for some species (9,17). The aim of this study was to develop a simple, rapid and easy methodology for DNA template preparation that could be used for PCR genetic studies on this pathogen, that grows slowly, sporulates on solid media, and has melanized spores and mature mycelia.

The consistency of PCR markers in all methodologies tested was verified in monosporic culture of C. lindemuthianum, obtained from single conidia isolated using a micromanipulator on an optical microscope. Different strains were used including a genetically modified hygromycin resistant construct; strain H2 (7).

Once isolated, single conidia were transferred individually to plates containing M3S liquid medium (10) and kept in the dark and at room temperature (19-20ºC) until the colony formed a fine mycelium that was suitable for testing (approximately 3 days). Initially PCR reactions were performed with DNA that was extracted and purified using traditional methods of 'mycelium maceration' in the presence of liquid nitrogen, extraction buffer and different methods of purification (6,10,13).

A second approach was to perform PCR reactions using protoplasts that were obtained by treating the mycelium with the enzymes NovozymeTM 234 (Interpex) or GlucanexTM (Novo Nordisk) or a mixture of Glucanase (Interpex) and Driselase (Interpex). The enzyme cocktail volume was twice as much as the volume of the filtered dried mycelium used for each digestion. To facilitate the isolation of protoplasts, combinations of different digestion times and temperatures were tested in SCE buffer (sorbitol 1M, sodium citrate 100 mM, EDTA 60 mM). The digestion times varied from 2 to 4 hours at intervals of 30 minutes with temperatures specified by the enzyme supplier. The enzyme concentration used was according to the product supplier. Afterwards, protoplasts were suspended in water, heated to 95ºC for 1 minute, centrifuged and the supernatant was used as template.

A third approach was based on precipitation and DNA isolation using ethanol 95% (2 volumes) and 10% sodium acetate (3M pH 5.2) in an ice bath. There followed a centrifugation step and the pellet was resuspended in ice-cold 70% ethanol. A further centrifugation step removed the last traces of ethanol and the pellet was air-dried. The DNA was dissolved in water (11) after protoplast production and incubation.

The fourth approach was collection, suspension and incubation of mycelial tips in reverse osmosis water (Millipore) for 3-5 minutes at high temperature (91ºC), followed by PCR. To perform the PCR reaction, the minimum possible amount of mycelium was taken using fine forceps (approximately 1mm2 of mycelium).

Different DNA primers were employed in this study (Table 1) designed from sequences known and deposited in Genbank. They were based on sequences encoding ribosomal DNA (internal transcribed spacer: its), for recognising the pAN7.1 (hphr or hph) hygromycin plasmid, for the endopolygalacturonase gene of C. lindemuthianum (clpg) and for the glyceraldehyde-3-phosphate dehydrogenase gene of C. lindemuthianum (clgpd). The total reaction volume was 25 µl containing 1.5 units Taq polymerase, 10x reaction buffer, MgCl2 at 25-50 mM, dNTPs at 10 mM, each primer at 10 pM and a variable quantity of DNA template depending on the concentration of the DNA in the sample, i.e. 0.5 µl of the plasmid was used compared to 2 to 4 µl of mycelial supernatant.

The conditions for the PCR reaction were denaturation at 91ºC for 30 seconds, annealing temperature (varied according to the primer used (Table 1) for 60 seconds; extension at 72ºC for 70 seconds and repeated for 35 cycles. Finally, a last cycle of 10 minutes at 72ºC was followed by holding at 4ºC. Amplification was carried out using a thermocycler of individual PCR minitubes (MJ Research Thermocycler - GRI). The PCR products were loaded into a 1.5-2.5% agarose gel, run at 100V for 30-60 minutes, stained with ethidium bromide (1 µg/ml) and visualized on a UV trans-illuminator.

Amplification occurred for all methodologies deployed including the fourth approach, when the incubation period was restricted to less than four minutes (Fig. 1a-b). However, when DNA was extracted and stored in Millipore water and frozen at -20ºC for over one month, the amplification products were not comparable with those obtained earlier, indicating that using fresh material was the best strategy (Fig. 1c).


With protoplasts, no differences were found in the results for different enzymes. However, Glucanex is currently promptly available and less expensive than the others. SCE (pH3-4) buffer was used for Glucanex for 3 hours at 37ºC at a concentration of 75 mg/ml.

The rapid amplification of small segments of DNA (<700 bp) was efficiently obtained using a small quantity of mycelium (the minimum possible) collected with a forceps and deposited directly in the minitube for PCR containing 5 µl of Millipore water. Simultaneously the PCR reaction mixture was prepared and kept cold in another tube, waiting for the template. The minitube containing mycelium was put in the thermocycler at 91ºC for 3 minutes, and then kept on ice. 4 µl of the supernatant was collected without centrifugation and used as a template in a 20 µl reaction mixture containing MgCl2 1.25 mM.

The PCR reaction performed directly on the mycelium was an efficient methodology to amplify DNA sequences smaller than 700bp. This result was demonstrated when primers (see Table 1 for details) hphr1-hphr6 (


Fig. 2a) and clpg1-clpg2 were used. A larger product than 700 bp was observed only on the reaction with pAN7-1 plasmid as template with the primer pair hph1-hph2 (Fig. 2b). However, the template copy number would have been much higher than from mycelial samples. In the case of primers its1-its4, its4-its5, its4-its6 where the product size was bigger than 700 bp, amplification occurred only when previously isolated DNA or protoplasts were used (Fig. 3a). A long incubation period for cells is likely to have led to DNA breakage resulting in non-amplification of longer segments of DNA (Fig. 1b, lines 4-6). On the other hand, an incubation time that was too short would result in insufficient DNA being released into the reaction mix.



The annealing temperature used for most reactions was 55ºC. In some cases, where the specific annealing temperature indicated by the enzyme's supplier was used, amplification did not occur if a high temperature had been previously used for incubation. The lack of amplification, however, may be due to the presence of inhibitors in the reaction, which did not allow the correct primer-template binding. It is supported by the observation that when the primers clpg1-clpg2 for DNA isolated at temperatures of 58ºC were used, amplification occurred normally (Fig. 3b). The quality of the template was such that increasing the annealing temperature gave sharper bands as the non-specific binding was reduced (Fig. 3b). When the annealing temperature below that indicated for each primer was used, the presence of different products was observed for some reactions, probably due to mis-priming (Fig. 1b, lanes 2-3).

With the hph1-hph2 primer pair at temperatures at or just above the Tm it is still possible to get specific annealing and good amplification if the template was of good quality and/or of high concentration. An example is with the plasmid where the relative concentration of template is high compared to the same target in the entire genome (Fig. 1b, Lane 1).

Using PCR methodology directly on mycelium is a rapid, practical and easy method that can be applied especially in the evaluation of a large number of samples and when the quantity of cells available is limited. However, the satisfactory results obtained here were only applicable to smaller amplicons. There is also variable intensity in PCR products that was expected because of the variable amount of DNA in the template. This methodology can be deployed successfully for genetic studies of other species but adaptations and/or modifications may be necessary because of cell wall composition, type of cell used.

Other methodologies have been developed such as PCR directly on spores without enzymes, using disruption by freezing and detergent (19), directly disrupting the spores (1), or by spore microwave treatment (9) and using enzymes for those products longer than 1kb (20). Here we present a simple, rapid and easy method for PCR-DNA extraction on young mycelia, with which it is possible to obtain a template in less than ten minutes. This method will allow the evaluation of a large number of samples in a relative short time allowing large scale screening of molecular markers for genetic characterization of C. lindemuthianum.

ACKNOWLEDGEMENT

We thank CAPES-Brazil for a scholarship (MGRM), Robert Cragg (University of Bath, UK) for his collaboration with enzymatic assays with Driselase, Dr. Pedro Sanchez (University of Bath, UK) for useful discussions, comments on the methodology, for plasmid pAN7-1 and for primers hph1-hph2, and Dr. Bernard Dumas (Université de Toulouse, France) for the H2 isolate. CAPES and FAPEMIG (Brazil) financed this project.

Submitted: January 24, 2002; Returned to authors for corrections: April 18, 2002; Approved: October 16, 2002

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  • *
    Corresponding author. Mailing Address: Departamento de Biologia. Universidade Federal de Lavras, Caixa Postal 37. 37200-000, Lavras, MG, Brasil. E-mail:
  • Publication Dates

    • Publication in this collection
      30 June 2003
    • Date of issue
      Apr 2003

    History

    • Accepted
      16 Oct 2002
    • Reviewed
      18 Apr 2002
    • Received
      24 Jan 2002
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