Accuracy of DNA amplification from archival hematological slides for use in genetic biomarker studies.
Archival slides are a potentially useful source of DNA for mutation analyses in large population-based studies. However, it is unknown whether specimen age or histological stains alter the accuracy of Taq polymerase or induce secondary mutations in sample DNA. To address this question, we evaluated five methods for extraction of genomic DNA from archival bone marrow slides of 17 leukemia patients and analyzed exons 1 and 2 of the N- and K-ras genes for the presence of mutations. Of the five methods, optimal DNA purification was achieved by boiling and phenol:chloroform extraction. N-and K-ras exons 1 and 2 were independently amplified using 35 cycles of PCR, and 6-12 clones for each exon were isolated and individually sequenced for each patient. Mutations were confirmed by repeat extraction, cloning, and sequencing. Sixteen of 17 patient samples were successfully amplified (94%), including slides up to 29 years old. Twelve slides had been stained with Wright-Giemsa, I stained with toluidine blue, and 4 were unstained. A total of 16 single-base mutations were identified of 33,840 nucleotides sequenced. No insertions or deletions were identified. Six of 16 single-base mutations were previously described activating mutations in codon 13 of N-ras exon 1. The 10 other mutations were in other regions of the N- and K-ras genes and were not reproduced after repeat extraction, cloning, and sequencing. The frequency of these other alterations was I of 3384 bp. This value is comparable with the inherent error frequency for Taq polymerase. Our findings suggest that high fidelity DNA amplification can be achieved using archival hematological slides as old as 29 years and can be reliably used in genetic analyses.
Boyle
E B
EB
Division of Pediatric Hematology, University of Minnesota Cancer Center, Minneapolis 55455, USA.
Steinbuch
M
M
Tekautz
T
T
Gutman
J R
JR
Robison
L L
LL
Perentesis
J P
JP
D001109:Archives; D001853:Bone Marrow; D002648:Child; D004247:DNA / Q000032:analysis*; D005819:Genetic Markers; D006801:Humans; D007938:Leukemia / Q000235:genetics*; D010865:Pilot Projects; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D013997:Time Factors; D014021:Tissue Preservation
D005819:Genetic Markers; D004247:DNA