Clinical application of a novel DNA fluorescence intercalator, pyrylium dye, for the quantification of PCR products.
Polymerase chain reaction (PCR) is a powerful tool for clinical diagnosis in the field of oncology, infection, and allergy. However, a simple and sensitive method for quantifying PCR products has not been established. We therefore used a novel fluorescence DNA intercalator, pyrylium iodide (P2) to quantify PCR products. A 838-bp fragment of the human beta-actin gene and a 374-bp fragment of the gene for ornithine decarboxylase (ODC) and ODC primer dimers were generated by PCR and quantified using either P2 or another fluorescence DNA intercalator, YOYO-1. In addition, utilizing RT-PCR and the P2 method, the ODC mRNA expression from 10 colonic cancers was quantified. Serially diluted beta-actin and ODC PCR products could be quantified using P2 without having to separate them from primer dimers and other components of the reaction mixture. However, YOYO-1 could not be used to quantify the PCR products because of high background fluorescence from the primer dimers. In the clinical study, ODC mRNA expression as quantified by P2 was significantly higher in cancerous tissue (113.8 +/- 4.1; mean +/- SEM; plate reader units) than in mucosa of normal appearance (68.4 +/- 4.8). P2 is a promising tool for quantifying PCR products.
Ichikawa
Y
Y
Second Department of Surgery, Yokohama City University School of Medicine, Japan.
Ishikawa
T
T
Momiyama
N
N
Chishima
T
T
Hasegawa
S
S
Yamaoka
H
H
Yamamoto
N
N
Okamoto
T
T
Kawaguchi
M
M
Shimada
H
H
D000199:Actins / Q000235:genetics; D000814:Aniline Compounds; D001583:Benzoxazoles; D003110:Colonic Neoplasms / Q000201:enzymology; D004247:DNA / Q000032:analysis*; D005456:Fluorescent Dyes; D006801:Humans; D007364:Intercalating Agents; D009955:Ornithine Decarboxylase / Q000235:genetics; D016133:Polymerase Chain Reaction; D011714:Pyrans; D011806:Quinolinium Compounds; D012333:RNA, Messenger / Q000378:metabolism
D000199:Actins; D000814:Aniline Compounds; D001583:Benzoxazoles; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D011714:Pyrans; D011806:Quinolinium Compounds; D012333:RNA, Messenger; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; C084408:2-methyl-4,6-bis(N,N-dimethylaminophenyl)pyrylium; D004247:DNA; D009955:Ornithine Decarboxylase