Molecular medicine I Flashcards
(53 cards)
What are the two steps in gene analysis?
Finding a correlation between a mutation and a disease
Detecting or predicting a disease based on knowledge obtained in the first phase
What is the direct method of mutation detection? What are its limitations?
Correlating a disease with a particular fragment of DNA
Limit = really time intensive (decades)
What is the indirect method of mutation detection? What are its limitations?
Following the inheritance of a marker. Marker may crossover (5% chance of this, thus test only 95% accurate)
What is the sanger method of DNA sequencing?
recopying DNA stands 4 times, adding labeled ACTor G adenine, then putting together like a jigsaw
What percent of the human genome actually has open reading frames?
3%
What percent of the genome varies between each person?
<1%
What are the two limits for sequencing?
Does not show on which chromosome mutation is
Will not show deletions or duplications of a chromosomal region
What does the term copy number variant refer to?
The fact that normal humans beings have small chromosomal insertions and deletions which (usually) do not have a major effect. (there are non diploid states of chromosomes)
What is comparative genome hybridization used for?
To determine the presence of CNVs
When is fluorescence in situ hybridization used?
To identify a chromosomal locus on a metaphase chromosome
What is hybridization?
Annealing of DNA strands together–utilized when you want to see if a patient has a DNA stand segment or not.
What is the polymerase chain reaction?
Using Polymerase to amplify the number of DNA strands based on the template strand of a patient.
(carried out by adding specific primers to block off region of interest, then changing temp)
How does PCR detect mutations?
- By adding primers that will only bind to mutants
2. seeing if primers deleted (large length change in DNA)
What is PCR used?
Qualitative testing of mutations
What technique would you use to get quantitative data on gene mutations?
Real-time PCR
What are the two independent factors in real time PCR?
Amount of template
number of PCR cycles
What is the dependent factors in real time PCR?
Amount of amplified DNA
What is reverse-transcriptase PCR used for?
Detection of RNA amounts (HIV load, expression of CA genes)
What does the enzyme reverse transcriptase do in reverse transcriptase PCR?
Synthesizes a DNA strand from RNA (rest of procedure follows PCR technique)
Why is it important that small oligonucleotides have very high sequence complementarity in Allele-specific oligonucleotide mutation detection?
means that they are extremely selective in what they bind to (will only bind to mutant/normal gene, and thus will cause only one spot on the plate to become positive)
What are DNA microarrays?
Plates that contain all known DNA defects that lead to disease. Can use the ASO concept to screen patients for genetic diseases.
How can CA cells be analyzed by ASOs?
Change the mRNA to cDNA by reverse transcriptase, using labeled nucleotides. Release onto ship with known DNA mutations.
What is malignant hyperthermia?
Mutation in Ryanodine receptor that causes the efflux of Ca in muscle cells, if there is halothane and succinyl-choline
What does warfarin inhibit?
Vitamin K epoxide reductase
