Suppose there is a genetic disorder caused by a single base change in a gene on...
Suppose there is a genetic disorder caused by a single base change in a gene on chromosome 11, which changes a cellular protein so that its function is altered. This mutation happens also to eliminate a cleavage site for a restriction endonuclease EcoRI (sequence: 5'-GAATTC-3'). If you isolated a portion of chromosome 11 DNA containing this defective gene, explain exactly how could you use restriction enzymes and gel electrophoresis to detect the base change. Please write 500 words or fewer.
Homework Answers
Procedure to identify mutation using restriction enzymes:
1. Design primers flanking the mutation
2. Perform PCR
3. Digest the PCR product with Eco RI
4. Run the products on an agarose gel
It is given that the mutation eliminates a site for Eco RI cleavage.
Since humans are diploid organisms, there are two copies of chromosomes.
Assume that the size of PCR product = 400 bp and there is only one Eco RI site in the sequence.
Since WT (+/+) individuals contain sequences that are cleaved by Eco RI in both chromosomes, they produce a single band upon digestion (200 bp).
Similarly, heterozygous individuals (+/-) produce two bands (400 bp and 200 bp) upon digestion.
Homozygous individuals produce a single band of 400 bp.
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