Nested amplification of COL2A1 3' variable region in skeletal remains.
We identified skeletal remains of a highly decomposed body by amplifying the COL2A1 3' variable region. We extracted degraded and contaminated DNA from teeth and bone fragments and amplified the hyper variable regions of COL2A1 by nested PCR. This method is 2,000 times more sensitive than the 27 cycle standard PCR. The sensitivity and specificity of the amplification was extremely improved using nested primer pairs. Using 47 cycles of total amplification by nested PCR, we were able to effectively amplify the target fragment. A total 47 cycles of dual PCR using one pair of primers was not as sensitive as nested PCR, which enabled us to amplify and detect the fragment from 5 pg of template DNA by agarose gel electrophoresis with ethidium bromide staining. We found that the hypersensitive nested PCR was suitable for the forensic identification of old, unidentifiable skeletal remains.
Honda
K
K
Department of Legal Medicine, Shinshu University School of Medicine, Nagano, Japan.
Sugiyama
E
E
Tsuchikane
A
A
Katsuyama
Y
Y
Harashima
N
N
Ota
M
M
Fukushima
H
H
D001483:Base Sequence; D001842:Bone and Bones / Q000737:chemistry*; D004247:DNA / Q000032:analysis*; D005554:Forensic Medicine; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic
D004247:DNA