DNA from processed and unprocessed wood: factors influencing the isolation success.
Molecular genetic markers have numerous potential applications in environmental forensics if DNA can be isolated from 'difficult' non-human biological material such as hairs, feathers, or wood. The identification of the origin of wood is particularly important in order to identify illegally harvested and traded timber and wood products. We describe success rates of DNA isolation from wood based on a simple, previously published extraction protocol. The protocol was used to isolate DNA from a total of 406 wood samples, mainly of the important tropical tree family Dipterocarpaceae. The reliability of the extraction method was confirmed by comparing fragment sizes and sequences after isolation of DNA from leaves and wood of the same trees. We observed the success of amplification of chloroplast DNA (cpDNA) fragments of different lengths by means of PCR, investigated key factors influencing PCR, and conducted inhibitor tests for a subset of the samples. The average rate of successful PCR amplification was 75.7%. Main factors influencing the success of PCR amplification were the size of the amplified fragment and the processing status of the wood. Short fragments and unprocessed wood resulted in higher success rates. The success rate was also dependent on the age (storage duration) of the wood probe and on the investigated species. Amplification success was higher if DNA was isolated from outer sapwood (without cambium) in comparison to DNA isolated from the transition zone between sapwood and heartwood and the inner heartwood. However, inhibitor tests also indicated more PCR inhibitory substances in the outer sapwood in comparison to transition wood and heartwood. The addition of polyvinylpyrolidone (PVP) to the lysis buffer proved to be highly efficient to improve the amplification success if inhibitory substances were present.
Buesgen Institute, Section of Forest Genetics and Forest Tree Breeding, University of Goettingen, Buesgenweg 2, 37077 Goettingen, Germany.
Yanti
Y
Rachmayanti
Ludger
L
Leinemann
Oliver
O
Gailing
Reiner
R
Finkeldey
D016172:DNA Fingerprinting / Q000379:methods; D018744:DNA, Plant / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D011205:Povidone / Q000737:chemistry; D014197:Trees / Q000235:genetics*; D014934:Wood / Q000235:genetics*
D018744:DNA, Plant; D011205:Povidone