I accidentally produce a mutant in my plant during I use an ordinary agrobacterium mediated transformation and I don't know any sequence for my mutant. So I am asking is there any PCR technique to differentiate between the heterzygous and the homozygous mutant?
1. First, you need to know whether this is a bona fide 'mutation'; some gene was knockout by your T-DNA, or was it just a tissue culture induced variation.
2. If it is a T-DNA insertion mutation, there are methods that you can use to figure out the sequence information neighboring the T-DNA. After you get the sequence neighboring the T-DNA, you can design specific primers for PCR to distinguish heterozygous and homozygous mutant genotypes.
1. First, you need to know whether this is a bona fide 'mutation'; some gene was knockout by your T-DNA, or was it just a tissue culture induced variation.
2. If it is a T-DNA insertion mutation, there are methods that you can use to figure out the sequence information neighboring the T-DNA. After you get the sequence neighboring the T-DNA, you can design specific primers for PCR to distinguish heterozygous and homozygous mutant genotypes.
Please also check out the similar discussion on RG: "Which is the best method for identification of transgene insertion sites in the plant genome?"
https://www.researchgate.net/post/Which_is_the_best_method_for_identification_of_transgene_insertion_sites_in_the_plant_genome
you can design primer for for mutant and wild sequence with forward and same reverse for each
u can go for heteroduplex pcr.
http://www.math.utah.edu/~palais/dnamath/DNAPapers/284.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC113844/
Finding mutation without prior information of the muation itself is difficult. You must first know the candidate location of the mutation before pcr and sequencing.
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