Molecular Medicine I Flashcards
What is molecular medicine?
The application of molecular biology, biochemistry, and genetics to the diagnosis and treatment of disease
Why is it so hard to identify a causative mutation?
Genome contains a lot of genetic variation:
About 1/1000 bp varies between individuals
Most variation does not cause disease
Sometimes genetic disease is not the result of sequence variation
Genome of the tumor cell is different from germline genome:
Tumor cells might have accumulated a lot of mutations that are not related to cancer
What does PCR stand for and do?
Polymerase Chain Reaction
Used to amplify DNA
How does PCR work?
Template DNA strands are separated, oligonucleotides bind to template
Oligo sequence needs to match template sequence
Polymerase extends oligonucleotides using the template
Cycle repeats - DNA between oligonucleotides is amplified exponetially
What can PCR tell you?
PCR requires pair of primers that binds to template DNA and then DNA polymerase amplifies DNA between primers.
Can detect point mutations - allele specific oligonucleotides, there is a mutation in the binding site and one primer does not bind so there is no amplification
Insertion/Deletion between primer sites - Different size of amplified DNA
What do allele-specific oligonucleotides arrays tell you?
Specificity of ASOs can be used to find variants
Design oligonucleotides that bind just to WT or mutant sequence - not to both
Fragment and label patient DNA and hybridize to ASO on slide
Detect label - hybridization to mutant ASO indicates presence of mutation
What does DNA sequencing do and what is the classical method?
Determines nucleotides sequence
Classical method is Di-deoxy chain termination method - sequence is read one DNA fragment at a time
What is another example of DNA sequencing?
Next generation sequencing (NGS)
What are you able to sequence?
Whole Genome Sequencing (WGS)
Whole Exome Sequencing (WES) - sequences only fraction of genome that is expressed
SNP typing - sequencing only bits of genome that are known to vary
How can methylation pattern be determined?
Bisulfite sequencing
Bisulfite treatment of DNA converts C to U
PCR converts U to T
Sequences and compare to treated and untreated DNA
What can you determine from bisulfite treatment?
Methylation pattern
Cytosine bases that were not converted to T were not methylated in the original sample
DNA methylation is tissue specific
What does FISH do and how?
Detect copy number variation
Hybridize labeled DNA probe to genomic DNA in interphase nucleus
Probe binds to a specific locus - there should be two fluorescent sites for autosomal loci
If there is 3 loci - region has 3 copies (trisomy)
If there is 1 locus - region has one copy (monosomy)
What can FISH be used in?
Use in cancer diagnosis and treatment
Detect amplification of growth factor receptor (HER2/neu)
What does multigene panel testing (MGPT) for hereditary cancer tell us?
Test determines the sequence of known oncogenes and tumor suppressors (10-30)
Test detects and classifies variation - pathogenic, likely pathogenic, uncertain significance, likely benign, benign
Data can predict risk, prognosis, and suggest treatment
Variants of uncertain significance - not enough information, not actionable
What is the testing strategy for hereditary cancer?
Test affected family member first to establish diagnosis and identify causative mutation
