Molecular Medicine Flashcards
DNA sequencing is most useful when?
—when the exact nature of the disease-causing mutation is known
Indirect Detection and Potential Problems w/ it
- -if exact mutation is not known one looks for presence of a linked marker
- -problem: marker may have separated from disease allele by recombination (if 10% recombination between marker and gene then one can only be 90% sure that a child without the marker is disease free)
Direction Detection and Potential Problems
- -if exact mutation is known, one can test Pt. DNA for it (basically sequence the DNA directly)
- -doesnt work if you dont know what mutation is since lots of SNP’s could show up as well
- -problem: Pt. might have a novel, rare or unknown mutation that is not detected
- -low penetrance means child w/ mutation might be healthy
What to sequence when considering sequencing?
- -not whole genome (not useful for clinical practice)
- -exome: all genome that is expressed 30 mil. bps
- -SNP typing: fewer than 10 mil. SNP’s in genome so to learn about individual characteristics of genome do SNP typing
- -only a few hundred SNP’s have been associated w/ disease and health risk
Copy Number Variations
- -presence of small chromosomal insertions or deletions
- -most are benign polymorphisms
Comparative Genome Hybridization
- -used to detect unbalanced chromosomal abnormalities
- -Pt. and control single stranded DNA labeled w/ flourescent dye and then hybridized to metaphase chromos to see how they match up
- -CNV evident by uneven labeling of the template chromsome i.e. excess of Pt. label fluorescence indicates a duplication in Pts. genome
FISH
- -fluorescence in-situ hybridization
- allows identification of specific chormosomal locus on a metaphase chromo by hybridization w/ a specific fluorescent probe
Hybridization of Nucleic Acids
- -denature Pt. DNA and attach to membrane
- -expose to bunch of labeled complimentary sequences and see what hybridizes
How is PCR used to detect genetic polymorphisms and mutations?
- -mutations can be detected w/ oligonucleotides that exlcusivley bind to either mutant or wildtype alleles (using mutant specific ASO’s as primers, only mutant alleles will be amplified)
- -Insertions or deletions between primer binding sites become obivous since you will get strands of different lengths amplified
Real-Time PCR
- -used for DNA quantification
- -amount of DNA amplified is measure at end of each cycle
- -determined how many cycles are required to reach a threshold
- –higher the number of template molecules = the fewer cycles needed to reach threshold
Reverse Transcriptase PCR
- -RNA —> DNA (by reverse transcriptase)
- -this cDNA serves as template for PCR
- -the more RNA there is = the more DNA will be amplified
Allele Specific Oligonucleotides (ASO’s) Tool
- -used to detect SNP’s and mutations
- -requires detailed knowledge about nucleotide sequence of gene under study
- -apply ASO spots for WT and mutant to slide
- -amplify Pt. DNA and label and hybridize to ASO spots
- -if both spots are labeled then Pt. is heterozygous and has both alleles
- -very specific method, ASO primers wont bind if one base is wrong
DNA microarrays
–basically just the ASO technique multiplied out and can be used for genetic screening of a Pt. for hundreds of known disease causing mutations
Analysis of gene expression by microarray
- -mRNA from Pt. and control is taken and reverse transcribed to ssDNA and labeled (Pt. red, control green)
- -then hybridized to microarray plate w/ oligonucleotides on them
- -DNA’s compete for binding
- -if certain gene is not expressed in a sample then there will be no ssDNA that will bind to that oligo and only the control DNA (green will bind)
Pharmacogenomics and polymorphism importance
- -polymorphisms in genes coding for drug targets determine how effective a drug inhibits the target enzyme
- -polymorphisms in drug metabolizing enzymes determine how fast a drug is cleared from system
- -goal is to find polymorphims that affect drug response in order to work out individual treatment plan