Rapid DNA extraction of pig ear tissues.
A single-step DNA isolation procedure from pig tissues was developed and the product used directly for polymerase chain reaction (PCR) amplification, single-strand conformational polymorphism (SSCP) analysis and sequencing. The procedure consists of proteinase K digestion of 2-10mg of fresh tissue, at 55 degrees C for 1h, followed by application of the products of digestion to filter paper. A 1.2mm-diameter punch of that paper has sufficient DNA to act as a template for PCR amplification. The quality of the genomic DNA appeared to be high as the PCR amplicons produced sharp banding patterns on both agarose gel electrophoresis and on SSCP analysis, and they could be used for DNA sequencing following cloning. The dried genomic DNA on filter paper can be kept at room temperature. The procedure is considered effective as it is simple, fast and inexpensive. It would be useful for large-scale genotyping and could be used to obtain genomic DNA from various tissues.
Kunhareang
S
S
Gene-Marker Laboratory, Department of Agricultural Sciences, Faculty of Agriculture and Life Sciences, P.O. Box 84, Lincoln University, Lincoln 7647, New Zealand.
Zhou
H
H
Hickford
J G H
JG
D000818:Animals; D004247:DNA / Q000302:isolation & purification*; D004423:Ear; D004587:Electrophoresis, Agar Gel; D019286:Endopeptidase K; D016678:Genome; D005838:Genotype; D009132:Muscles; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*; D012867:Skin; D013552:Swine / Q000235:genetics
D004247:DNA; D019286:Endopeptidase K