Modified inter-simple sequence repeat PCR protocol for use in conjunction with the LI-COR Gene ImagIR2 DNA analyzer.
Inter-simple sequence repeat (inter-SSR) PCR was assessed for use in variety testing of chrysanthemum. This method was modified to allow detailed analysis of DNA profiles on a LI-COR Gene ImagIR2 DNA analyzer system. Protocols for unlabeled PCR were unsuccessful in producing labeled products when using infrared (IR) dye-labeled primers. Various modifications to the known protocols were investigated: (i) different ratios of labeled to unlabeled primer; (ii) various annealing temperatures; (iii) the use of an IR genotyping kit; (iv) end labeling; and (v) direct incorporation and cycle labeling. Successful amplification using labeled primers only occurred when two consecutive reactions were performed. The first PCR was performed using standard protocols for unlabeled reactions. The second PCR used a dilution of these reaction products as a template and 50% IR-labeled and unlabeled primer. The complete procedure leading to a high-resolution analysis of inter-SSR PCR products on a LI-COR system is reported for the first time. This system allows high-throughput fingerprinting with the potential for applications on a commercial scale.
Jackson
J A
JA
National Institute of Agricultural Botany, Cambridge, UK. jenny.jackson@niab.com
Matthews
D
D
D011723:Chrysanthemum cinerariifolium / Q000235:genetics; D004396:Coloring Agents; D017931:DNA Primers; D018744:DNA, Plant / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D007259:Infrared Rays; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods*; D012984:Software
D004396:Coloring Agents; D017931:DNA Primers; D018744:DNA, Plant