Comparison of five DNA quantification methods.
Six commercial preparations of human genomic DNA were quantified using five quantification methods: UV spectrometry, SYBR-Green dye staining, slot blot hybridization with the probe D17Z1, Quantifiler Human DNA Quantification kit and RB1 rt-PCR. All methods measured higher DNA concentrations than expected based on the information by the manufacturers. UV spectrometry, SYBR-Green dye staining, slot blot and RB1 rt-PCR gave 39, 27, 11 and 12%, respectively, higher concentrations than expected based on the manufacturers' information. The DNA preparations were quantified using the Quantifiler Human DNA Quantification kit in two experiments. The measured DNA concentrations with Quantifiler were 125 and 160% higher than expected based on the manufacturers' information. When the Quantifiler human DNA standard (Raji cell line) was replaced by the commercial human DNA preparation G147A (Promega) to generate the DNA standard curve in the Quantifiler Human DNA Quantification kit, the DNA quantification results of the human DNA preparations were 31% higher than expected based on the manufacturers' information. The results indicate a calibration problem with the Quantifiler human DNA standard for its use with the Quantifiler Human DNA Quantification kit. The possible reasons for the problem are discussed and a solution is suggested. The results emphasise the need for standard reference DNA material and standard methods for DNA quantification.
Nielsen
Karsten
K
Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen, Denmark. karsten.nielsen@forensic.ku.dk
Mogensen
Helle Smidt
HS
Hedman
Johannes
J
Niederstätter
Harald
H
Parson
Walther
W
Morling
Niels
N
D001774:Blood Chemical Analysis; D002138:Calibration; D045744:Cell Line, Tumor; D002886:Chromosomes, Human, Pair 17 / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D053803:Forensic Genetics; D006801:Humans; D008223:Lymphoma / Q000378:metabolism / Q000473:pathology; D008297:Male; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009930:Organic Chemicals / Q000378:metabolism*; D010920:Placenta / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D012015:Reference Standards; D013056:Spectrophotometry, Ultraviolet
D005456:Fluorescent Dyes; D009930:Organic Chemicals; D004247:DNA