Related Experiment Video
Updated: Jul 10, 2026
07:04
Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients
Published on: February 11, 2017
One multiplex control for 29 cystic fibrosis mutations.
Roger V Lebo1, Michelle Bixler, Donna Galehouse
1Department of Pathology, Akron Children's Hospital, Akron, OH 4430-1062, USA. rlebo@chmca.org
Genetic Testing
|October 24, 2007
Summary
A novel method synthesizes an unlimited supply of genetic controls for multiplex mutation assays. This approach ensures accurate detection of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in patient samples.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Multiplex mutation assays require reliable controls for accurate results.
- Existing methods for generating genetic controls are often limited in scope and supply.
Purpose of the Study:
- To develop a simple and scalable method for synthesizing an inexhaustible supply of homozygous and heterozygous genetic controls.
- To validate this approach for controlling multiplex mutation assays, specifically for cystic fibrosis transmembrane conductance regulator (CFTR) mutations.
Main Methods:
- Synthesizing and cloning mutant gene fragments containing up to four homozygous mutations.
- Incorporating 29 cystic fibrosis transmembrane conductance regulator (CFTR) mutations into 17 gene fragments using PCR and mutagenic primers.
- Cloning these fragments into plasmid vectors and sequencing for verification.
- Diluting cloned fragments with yeast genomic DNA to mimic human genome equivalents for use as a positive control in PCR reactions.
Main Results:
- Successfully created a single control mixture containing 29 cystic fibrosis transmembrane conductance regulator (CFTR) mutations.
- Validated the control in over 200 multiplex clinical polymerase chain reaction (PCR) analyses involving more than 4,000 patient samples.
- Demonstrated simultaneous control for PCR amplification and substrate specificity without cross-contamination.
Conclusions:
- The described approach provides a robust and adaptable method for generating a comprehensive and inexhaustible supply of genetic controls for multiplex mutation assays.
- This clinically validated control can be readily modified for various test formats, offering a superior alternative to rotating human genomic controls.
More Related Videos
Related Concept Videos
01:24Cystic Fibrosis: Management
Cystic fibrosis (CF) is an autosomal recessive disorder that predominantly affects individuals of Northern European descent, occurring at a rate of 1 in 3500. It is caused by a genetic mutation in a gene on chromosome 7, most commonly the ΔF508 mutation, that codes for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. This results in thicker mucus secretions and obstruction pathologies in multiple organs, including the lungs and sinuses.
Sinus disease and chronic sinusitis...
Sinus disease and chronic sinusitis...
01:23Cystic Fibrosis: Pathogenesis
Cystic fibrosis (CF), an autosomal recessive disorder, significantly affects the function of exocrine glands. This genetically inherited disease is characterized by the production of thick and sticky mucus, which can severely affect various organs and systems in the body.
CF is primarily caused by a genetic mutation in a chromosome 7 gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most common gene mutation leading to CF is the ΔF508 mutation, but...
CF is primarily caused by a genetic mutation in a chromosome 7 gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most common gene mutation leading to CF is the ΔF508 mutation, but...