Recombinant DNA Technology And Biotechnology 11-21 Flashcards
Cystic fibrosis is caused by a ________ mutation
Genetic
Cystic fibrosis results in defective what
Cl ion channeling
What causes defective Cl ion channeling in cystic fibrosis
Due to improper folding and assembly of CFTR-cystic fibrosis transmembrane conductance regulator-chloride transport protein prematurely degraded
What is the most common lethal genetic mutation in caucasians?
Cystic fibrosis
Clinical diagnostic hallmark of cystic fibrosis
Elevated chloride levels in sweat- specific to CF
Cystic fibrosis affect on lungs
Lack of chloride secretion leads to dehydration of the mucus, mucus accumulates in the lungs, obstructs airway passages, predisposed to recurrent lung infections
Other symptoms of cystic fibrosis
Also causes pancreatic enzyme insufficiency and poor nutrition among other problems
What are most cases of cystic fibrosis due to?
A 3 nucleotide deletion in the CTFR gene that leads to a missing phenylalanine residue
Detection and diagnosis of common CF mutation
Using ASO probes (allele specific oligo
PCR and cystic fibrosis
PCR using primers specific to the region of the deletion-PCR product is only a portion of the gene
-other known mutations causing CF would require different analysis
Sickle cell anemia and cystic fibrosis genetic diagnosis
Very specific mutations-single nucleotide change in sickle cell anemia and 3 nucleotide deletion in CF
USE ASO PROBES AND PCR
What types of tests would you do for sickle cell anemia and cystic fibrosis
ASO probes and PCR
PKU
All newborns are screened for PKU-blood test for elevated phenylalanine levels and low levels of tyrosine
-400 different known mutations
Where can mutations occur in PKU
In any of the 13 exons
What type of mutations are PKU
Majority are missense, some splice, nonsense, insertion, and deletion mutations
How big are introns for PKU
HUGE
To do a genetic screen for PKU we must have:
- The DNA from several family members including an affected individual
- An identifiable marker associated (linked) with the particular mutation in this family (an RFLP)
What would the marker be for PKU?
Polymorphisms (nucleotide change that results in an added or lost restriction site)
-generally the marker is not actually the disease causing mutation
Markers are at least _____ accurate in predicting the presence of a mutation
98%
What happens when a marker is identified in PKU?
A genetic screen can be performed (determine if a fetus has it)
If a couple has one affected child and two phenotypically normal children, both parents are ______ carriers.
Obligate. Neither parent expresses it
Myotonic dystrophy
- trinucleotide repeat expansion disease in the 3’ non-coding region of a proline kinase gene
- no phosphatase, cant relax muscles
What is the most common form of adult muscular dystrophy?
Myotonic dystrophy
Because myotonic dystrophy is associated with repeat expansion, it will produce _____ when digested with a restriction enzyme on both sides of the expansion
RFLPs
What happens to expansions as you go though generations?
One mutant allele increases in size in subsequent generations (correlates to severity of disease)
____ can be used to diagnose triplet repeat expansions
PCR
Why can you not use PCR for myotonic dystrophy?
The repeat can be so huge that PCR is difficult
Current methodology for testing for HIV uses __________ for diagnosis of HIV exposure. Why?
- immunoassay
- after exposure to HIV the body produces antibodies to HIV, immunoassay use enzymes linked antibody that recognize HIV antibodies
What two tests do you use to detect HIV?
- ELISA
2. Western blot
Why do we need to do western blot after ELISA for HIV?
ELSA is so sensitive that it can produce false positives.
What do immunoassay using antibodies to HIV-antibody require
That the body has mounted an immune response to a detectable level- requires a certain incubation time (6 months)
Can use ______ for HIV provirus to test immediately
PCR
__________ is used to monitor viral load in HIV positive patients
Quantitative PCR
What is tested in paternity testing?
VNTRs
What test do you do for paternity testing
Usually done using PCR
Legal accuracy of paternity tests
Differs in different states, paternity index is a measure of accuracy
Why do paternity cases always require several different VNTRs to be used?
Different VNTRs have a different paternity index
Testing forensics
Restriction digests, southern blot with probe for tandem repeats (VNTR)
Pattern of ______ can be used as a molecular fingerprint in forensics
RFLPs (VNTRs)
-normally several restriction digests and several different probes would be tested
What is a good method if the amount of DNA evidence is too small to do southern blotting? (Forensics)
PCR based approaches
