Day 3, Lecture 1 (Aug 24) Genetics III: Recombinant DNA Technology and DNA Mutation Flashcards
Medical Applications of DNA Technology
- Identification of disease-causing genes
- Molecular Genetic (DNA) Diagnosis
- Genetic diagnosis of infectious disease (such as Tuberculosis, HIV)
- Synthesis of recombinant proteins for therapies (e.g. insulin, growth hormones)
- Forensic applications (DNA fingerprinting)
- Gene therapy
Procedure of DNA cloning
A DNA fragment is inserted into a vector to create a recombinant vector. A vector is a replicon that makes multiple copies of itself (clones) when introduced into a host bacterial cell ( transformation)
Polymerase Chain Reaction (PCR)
- In vitro method for amplifying DNA
- Over million-fold amplification in about 2 hours, starting from a small quantity of input DNA
- DNA is used for further analysis/manipulation as with cloned DNA
- Replaced cloning as the preferred method for amplifying DNA
Reverse Transcription- Polymerase Chain Reaction (PCR)
Medical applications of PCR
- Advantages
- Identification of minute quantities of DNA
- Detection of specific DNA sequences
- Mutation detection (molecular diagnosis)
- DNA sequencing (molecular diagnosis)
- DNA fingerprinting (identity/paternity testing)
- Diagnosis of infectious diseases (TB, HIV)
- RT-PCR:
- Detection of splice-site mutations convenient mutation detection (no introns)
Missense Mutation
- Replaces one a.a. codon for another
Nonsense mutatation
Replaces an a.a. codon for a stop codon
Splice-site mutation
Creates/destroys splice sites
Classification of DNA mutations
In-frame deletion vs. Out-of-frame deletion
Premature STOP codons induce ______
- nonsense mediated RNA decay
Explain Splice Site mutations
Diseases caused by triplet repeat expansion
- Note that sense these are naturally occurring repeats once there is an occurence you can have anticipation and the occurence of a genetic disorder at progressivley earlier age in successive generations is likely
- Disease severity is proportional to the length of the repeat expansion
- Expanded repeats are unstable and they may increase in length from one generation to the next
What is the gold standard for confirmation of mutations
Sanger’s DNA seqencing
Most mutations arise due to _____
- replication errors
Causes of mutations
The ___ dinucleotide is a mutation hotspot
- CpG dinucleotide
- mutation hotspot with 8.5 times greater mutation rate over other dinucleotides
Nucleotide excision repair
- repair for UV-induced damage
Mutator phenotype
Defects in DNA repair pathways cause acculation of multiple mutations
____ is a common consequence of defective DNA repair
Cancer
DNA Repair mechanisms
Unequal recombination causes
- deletions/duplications
- Note flanking of low copy repeats can lead to this miss pairing and thus lead to duplications and deletions
Chromosome 15 has an area that is greatly effected by unequal recombination causing deletions/duplications thus leading to Pader Willi Syndrome and Angelman Syndrome. What is this area flanked by?
low copy repeats of the HERC2 gene
restriction enzyme
an enzyme produced chiefly by certain bacteria, having the property of cleaving DNA molecules at or near a specific sequence of bases.
cloning vector
A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.
Southern blot
Northern blot
Mutagenesis
is a process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens
