3. You are working in a lab that is studying Disease X, a genetic disorder that
ID: 131982 • Letter: 3
Question
3. You are working in a lab that is studying Disease X, a genetic disorder that arises from a trinucleotide repeat expansion mutation, a mutation where entire codons are multiplied during the replication process. The mutation is found in the CaM gene, which is a 450-base pair (bp) sequence. The trinucleotide repeats expansion results in the addition of 10 codons (30 bp) at nucleotide 150 (ie in the centre of the gene, away from the 5' and 3' end). The sequence of the normal gene is shown below ATGGCTGATCAGCTGACCGAAGAACAGATTGCTGAATTCAAGGAAGCCTTCTCCCTATTTGATAAAGATG GCGATGGCACCATCACAACAAAGGAACTTGGAACTGICATGAGGTCACTGGGTCAGAACCCAACAGAAGC TGAATTGCAGGATATGATCAATGAAGTGGATGCTGATGGTAATGGCACCATTGACTTCCCCGAATTTTTG ACTATGATGGCTAGAAAAATGAAAGATACAGATAGTGAAGAAGAAATCCGTGAGGCATTCCGAGTCTTTG ACAAGGATGGCAATGGTTATATCAGTGCAGCAGAACTACGTCACGTCATGACAAACTTAG AACAGATGAAGAAGTAGATGAAATGATCAGAGAAGCAGATATTGATGGAGACGGACAAGTCAACTATGAA GAATTCGTACAGATGATGACTGCAAAATGA Design primers for the amplification of this gene (4 marks) Design a PCR-based method to detect this disorder in patients. (4 marks) Describe or draw your expected results for an individual with the disease vs. a healthy individual control. (2 marks) A sample collected from a patient with similar symptoms to those suffering from Disease X has been brought to the lab. You use your PCR based test and determine that the CaM gene is the same size as that of a healthy individual. You suspect that the arises from a point mutation (a single nucleotide change that results in a defect in the protein.) Describe a how you could confirm this hypothesis. (6 marks) a. b. c. d.Explanation / Answer
a. One needs to use a forward and a reverse primers for its amplification.
The primers can be as follows:
Forward primer : 5' ATTGGCTGATCAGCTGAC 3'
Reverse primer: 5'TCATTTTCAGGTCATC3'. One should however bioinformatically look at the theoretical annealing temperature, melting temperature as well as formation of dimers and hair pin structures.
b. One can perform a PCR, do a PCR based purification of specific gene or excise the band from an agarose gel after PCR and send it for sequencing to confirm the disorder. One can also perform RFLP based detection of disorder.
c. A healthy individual would not have got the trinucleotide repeat of 10 codons (30 bp), instead would lead to a single codon at that particular region.
d. The CaM gene can be first amplified, sequenced and then compared using the parent gene as the template. If one finds a point mutation in the sequenced gene, one should first determine it functionally as well. An individual needs to perform a functional test as well (in terms of signaling, calcium binding, localisation etc). This depends upon the property of the gene as well as protein.
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