title: Real-Time PCR Quantification of Chloroplast DNA Supports DNA Barcoding of Plant Species.
abstract: Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.
authors.affiliation: National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba, 277-0882, Japan. kikkawa@nrips.go.jp.
authors.forename: Hitomi S
authors.initials: HS
authors.lastname: Kikkawa
authors.affiliation: National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba, 277-0882, Japan.
authors.forename: Kouichiro
authors.initials: K
authors.lastname: Tsuge
authors.affiliation: National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba, 277-0882, Japan.
authors.forename: Ritsuko
authors.initials: R
authors.lastname: Sugita
mesh_terms: D052160:Benzothiazoles; D058893:DNA Barcoding, Taxonomic / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D018742:DNA, Chloroplast / Q000032:analysis* / Q000737:chemistry / Q000145:classification; D003959:Diamines; D009930:Organic Chemicals / Q000737:chemistry; D010944:Plants / Q000145:classification / Q000235:genetics; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D018742:DNA, Chloroplast; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I
keywords: Botanical samples; DNA barcoding; Quantitative real-time PCR; Ribulose bisphosphate carboxylase large subunit gene