Question

Case Study 12-21 A 7-month-old child suffered from sleep apnea and difficulty breathing. Respiratory infections and allergies were ruled out. From the family history, the physician suspected congenital congestive hypoventilation syndrome (CCHS). CCHS results, in part, from abnormal function of the PHOX2B gene product caused by a triplet-repeat expansion. The expansion of the triplet GCN in the DNA sequence (N is any base) results in the insertion of additional alanines in the Phox2B protein. The symptoms of the syndrome include interruptions in breathing during sleep and other abnormalities in the autonomic nervous system. A blood sample from the patient was tested for the GCN expansion by PCR using primers that flanked the location of the potential expansion. The amplicons were resolved by agarose gel electrophoresis. <Insert Fig 12-26> gcagcagcagcggeggcggccgeggcagoggcggcggcggcageggcagoggcggcaget PHOX2b exon 3 4 5 6 7 8 9 10 11 12 PAGE 22 20 - (Normal) D1 400- 300 200- (Normal) Agarose (rapid test) a. 1. How will an expansion affect the PCR product in this test? It will have no effect; the product must be probed to detect the expansion. b. The PCR product will be smaller than if no expansion were present. c. The expansion will increase the size of the PCR product. d. There will be no PCR product because the expansion will be too large to amplify efficiently.

Answer 1

c. The expansion will increase the size of the PCR product.

The length of the PCR product depends on the nucleotides present in the template sequence. If the template has a triplet-repeat expansion, it would increase the length of the template sequence to be amplified. Let us assume the triplet is repeated 10 times, the gene would be increased by 30 nucleotides. This change can be seen in agaraose gel electrophoresis. Compared to the normal gene template, the abnormal gene template would give a larger PCR product.