1. Putting it all together: Finding the cystic fibrosis gene Cystic fibrosis (CF) is a genetic disorder that is relatively common in some ethnic groups A defective gene causes the body to produce an abnormally thick, sticky mucus that clogs the lungs and leads to life-threatening lung infections. These thick secretions also obstruct the pancreas, preventing digestive enzymes from reaching the intestines to help break down and absorb food. The mucus also can block the bile duct in the liver, eventually causing permanent liver damage in approximately six percent of people with CF. CF occurs in approximately one of every 3,500 live births.

2. Putting it all together: Finding the cystic fibrosis gene What did we know before getting started? –CF is inherited in a classically Mendelian fashion –More than 10 million Americans are unknowing, symptomless carriers of the defective CF gene. –An individual must inherit two defective CF genes— one from each parent—to have CF. Each time two carriers conceive, there is a 25 percent chance that their child will have CF; a 50 percent chance that the child will be a carrier of the CF gene; and a 25 percent chance that the child will be a non-carrier.

3. Putting it all together: Finding the cystic fibrosis gene Finding the gene is the first step in treating it. Once the gene is found, we can determine the nature of the allele that causes the disease and, potentially, treat the problem. In addition, once the gene is found, simple tests for determining the genotype of an individual would aid in diagnosis and assessment of reproductive risks. Finding the gene took 4 years and was largely based on linkage analysis (this was before the human genome project, the mid- 1980s).

4. Putting it all together: Finding the cystic fibrosis gene Step 1: A large number of families were screened for genetic markers known as RFLPs –RFLP – restriction fragment length polymorphism, sequences in the genome that are cut by a restriction enzyme in some people but not in others –See p. 328 in text and lecture from chapter 10 Step 2: Determine which, if any, RFLPs might be linked to the CF gene –Use pedigrees to determine if any RFLPs are co- inherited with CF –See p. 682-683 and lecture for chapter 20

5. Putting it all together: Finding the cystic fibrosis gene Step 3: One RFLP was found to be loosely linked to CF –This suggests that the RFLP is located relatively close to the CF gene –The RFLP was isolated to chromosome 7 via in situ hybridization –See p. 340 in text and lecture from chapter 10 Step 4: Additional analysis confirmed two more RFLPs that were tightly linked to the CF locus –MET and D7S8 –Narrowed the region down to ~1.5 million bp

6. Putting it all together: Finding the cystic fibrosis gene Step 5: Generate a large-insert DNA library of the region –Use rare-cutting restriction enzymes to generate large fragments of the genome –See p. 328 in text and lecture from chapter 10 –Clone the large fragments into an appropriate vector –See p. 341-345 and lecture from chapter 10 Step 6: Screen the library (using hybridization) to determine clones that have the DNA from the region of interest –See p. 340 in text and lecture from chapter 10 Step 7: Further analysis using additional probes and linkage analysis narrowed the region down to ~500,000 bp

7. Step 8: Chromosome jumping – a combination of hybridization probing and DNA sequencing –Cut genomic DNA from the region of interest –Circularize the DNA using ligase –Cut the circularized fragment again and sequence the ‘ends’ –Use the sequenced ends as probes to determine which end is closer to the target –Repeat until you arrive at the target Putting it all together: Finding the cystic fibrosis gene

8. How do you know when you’ve arrived at the target? Nobody knew what the sequence of the gene was

9. Step 9: Determine the when you have gotten gene sequence –Hybridize probe sequences to DNA from other organisms (Southern blot, p. 338) –Sequences that contain coding DNA will hybridize while non-coding DNA will not –Why? Coding DNA changes more slowly than non- coding DNA –See chapter 9 lecture, figure 9-19, and p. 316 Putting it all together: Finding the cystic fibrosis gene

10. Step 10: Determine if the DNA sequences that are highly conserved are expressed in the cells affected by CF –Hybridize probe sequences to mRNA/cDNA from cultured cells (lung, sweat glands, pancreas, brain, heart, etc) (Northern blot, p. 338) –Cell types that are affected by CF would be expected to express the CF gene. Cell types that are not affected by CF shouldn’t –Cell types that are expressing the CF gene hybridize with the cDNA Putting it all together: Finding the cystic fibrosis gene

11. Step 11: Sequence the entire region and characterize the gene in individuals with and without the disease –Determine exon and intron locations and predict the amino acid sequence of the gene –The gene is now called the cystic fibrosis trans- membrane conductance regulator (CFTR) –This just requires sequencing the rest of your clones from the region and standard sequencing protocols (see pp. 331-333) –The gene contains 24 exons and spans ~280 kb. –The final amino acid product is 1480 aa long Putting it all together: Finding the cystic fibrosis gene

12. Step 11: Sequence the entire region and characterize the gene in individuals with and without the disease –The most commone CF causing mutation is a three base pair deletion in the gene of people who are carriers or affected –The amino acid phenylalanine is deleted from the amino acid chain –Other mutations also exist but are not as common Putting it all together: Finding the cystic fibrosis gene

13. Testing for CF is easy using modern techniques PCR followed by a hybridization technique known as an oligonucleotide ligation assay (OLA) Putting it all together: Finding the cystic fibrosis gene