Reliable genotyping of samples with very low DNA quantities using PCR.
Our purpose was to identify an experimental procedure using PCR that provides a reliable genotype at a microsatellite locus using only a few picograms of template DNA. Under these circumstances, it is possible (i) that one allele of a heterozygous individual will not be detected and (ii) that PCR-generated alleles or 'false alleles' will arise. A mathematical model has been developed to account for stochastic events when pipetting template DNA in a very dilute DNA extract and computer simulations have been performed. Laboratory experiments were also carried out using DNA extracted from a bear feces sample to determine if experimental results correlate with the mathematical model. The results of 150 typing experiments are consistent with the proposed model. Based on this model and the level of observed false alleles, an experimental procedure using the multiple tubes approach is proposed to obtain reliable genotypes with a confidence level of 99%. This multiple tubes procedure should be systematically used when genotyping nuclear loci of ancient or forensic samples, museum specimens and hair or feces of free ranging animals.
Taberlet
P
P
Laboratoire de Biologie des Populations d'Altitude, CNRS UMR 5553, Université Joseph Fourier, BP53, Grenoble, France.
Griffin
S
S
Goossens
B
B
Questiau
S
S
Manceau
V
V
Escaravage
N
N
Waits
L P
LP
Bouvet
J
J
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D003198:Computer Simulation; D005243:Feces / Q000737:chemistry; D005838:Genotype; D017408:Guidelines as Topic; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D001503:Ursidae / Q000235:genetics