Abstract: The products obtained by long range PCR based on objective sequences were the effective probes for FISH to carry out molecular cytogenetic study.However,it is very difficult to amplify more than 5 kb-long fragments effectively by traditional PCR techniques.Suitable reaction conditions and reaction system are the necessary prerequisite for running effective LR-PCR amplification.In order to obtain LR-PCR products based on the objective sequences as FISH probes,80 pairs of LR-PCR primers were designed based on the sequences of repeat-free regions on the top of Chinese cabbage chromosome A03,and PCR technique system was optimized from the following aspects containing the template quality of Chinese cabbage genomic DNA,dNTPs concentration,annealing temperature and extended time.Results indicated that genomic DNA of seedling leaves and LA Taq polymerase enzyme could improve the amplification quality and efficiency of long-range PCR.And 5-15 kb PCR products could be amplified by the reaction system with 20 μL of total volume,comprising of 50 ng/μL DNA 2 μL,2.5 mmol/L dNTPs 1.6 μL,10 μmol/μL of each primer 1 μL,10×LA PCR Buffer Ⅱ (including Mg²⁺) 2 μL,5 U/μL LA Taq enzyme 0.2 μL.PCR reaction procedure was followed as:98℃ for 15 s;58-64℃ for 10 s,68℃ for 5 min,35 cycles;final extension at 68℃ for 10 min kept at 4℃.Under these conditions,60 fragments with 5-15 kb length were obtained from Chinese cabbage genome.This research would establish the basis for carrying out long range PCR-FISH on meiotic pachytene chromosomes of Chinese cabbage and for clarifying evolutionary by comparing chromosome painting among close related species.

Key words: Chinese cabbage, Long range PCR(LR-PCR), System optimization, Single copy sequences

CLC Number: 

• Q78

• S634.03