Transcript Putting it all together: Finding the cystic fibrosis gene

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.
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.
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).
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 coinherited with CF
– See p. 682-683 and lecture for chapter 20
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
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
Putting it all together:
Finding the cystic fibrosis gene
• 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
• How do you know when you’ve arrived at
the target? Nobody knew what the
sequence of the gene was
Putting it all together:
Finding the cystic fibrosis gene
• 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 noncoding DNA
– See chapter 9 lecture, figure 9-19, and p. 316
Putting it all together:
Finding the cystic fibrosis gene
• 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
• 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 transmembrane 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
• 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
• Testing for CF is easy using modern techniques
• PCR followed by a hybridization technique
known as an oligonucleotide ligation assay
(OLA)