Evaluation of DNA extraction methods of rumen microbial populations.
The dynamism of microbial populations in the rumen has been studied with molecular methods that analyze single nucleotide polymorphisms of ribosomal RNA gene fragments (rDNA). Therefore DNA of good quality is needed for this kind of analysis. In this work we report the evaluation of four DNA extraction protocols (mechanical lysis or chemical lysis with CTAB, ethylxanthogenate or DNAzol(®)) from ruminal fluid. The suitability of two of these protocols (mechanical lysis and DNAzol(®)) was tested on single-strand conformation polymorphism (SSCP) of rDNA of rumen microbial populations. DNAzol(®) was a simple method that rendered good integrity, yield and purity. With this method, subtle changes in protozoan populations were detected in young bulls fed with slightly different formulations of a supplement of multinutritional blocks of molasses and urea. Sequences related to Eudiplodinium maggi and a non-cultured Entodiniomorphid similar to Entodinium caudatum, were related to major fluctuating populations in an SSCP assay.
Centro Multidisciplinario de Estudios en Biotecnología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Michoacana de San Nicolás de Hidalgo, Km 9.5 carretera Morelia-Zinapécuaro, La Palma, C.P. 58893 Tarímbaro, Michoacán, Mexico.
Gabriela
G
Villegas-Rivera
Yevani
Y
Vargas-Cabrera
Napoleón
N
González-Silva
Florentino
F
Aguilera-García
Ernestina
E
Gutiérrez-Vázquez
Alejandro
A
Bravo-Patiño
Marcos
M
Cajero-Juárez
Víctor Manuel
VM
Baizabal-Aguirre
Juan José
JJ
Valdez-Alarcón
D053000:Analytic Sample Preparation Methods / Q000379:methods*; D000818:Animals; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002417:Cattle; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification*; D008297:Male; D008969:Molecular Sequence Data; D010802:Phylogeny; D018807:Polymorphism, Single-Stranded Conformational; D012417:Rumen / Q000382:microbiology*
D004269:DNA, Bacterial; D004275:DNA, Ribosomal