Flashcards in Molecular medicine I Deck (53):
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?
What percent of the genome varies between each person?
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?
1. 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?
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
What are the two sources of possible mutations that can change the way warfarin is degraded?
Polymorphism in CYP2C9 (codes for p450) --> lower tolerance
Mutation in subunit of vitamin K expoxide reductase (VKORC1) --> increased tolerance
What are the two ways to collect a sample from a fetus for genetic testing?
Amniocentesis (15/16 weeks withdrawn amniotic fluid)
Chorionic villus sampling (cells from chorion taken at 10-12 weeks)
Why is chorionic villus sampling riskier than amniocentesis? What is the advantage?
Risky b/c chance of missing mosacism
Able to do it earlier
How are epigenetic changes analyzed?
minute amount of DNA treat methylated DNA with chemicals
How are proteins detected and quantified?
The antibodies used in protein detection can do what three things?
Detect presence of antigen
localize antigens in vivo
What type of cells produce antibodies?
What is the area of the antigen that antibodies bind to?
How many types of antibodies can B cells produce?
What are monoclonal antibodies? What are polyclonal antibodies?
Polyclonal = "Antibodies that are secreted by different B cell lineages within the body (whereas monoclonal antibodies come from a single cell lineage)": -Wikipedia
What is the sandwich model of ELISA?
Antibodies for protein on bottom
Antigen/protein in the middle
Receptor molecule on top
What is the sandwich model of ELISA used for?
Quantification of proteins
(PTH, HbA1c, CRP)
What is the indirect model of ELISA?
Anitgen linked to bottom.
General antibodies with receptor added.
What is the indirect model of ELISA used for?
HIV and autoimmune diseases
What protein detection technique would you use to test for RA? What is the antigen in this case?
Indirect ELISA--immunoglobin G
What is western blotting used for?
Indicated molecular weight
What are the two specific used for Western blotting in the handout?
Confirm HIV (Need two tests)
Diagnose parasite infections
What is the sample used, and what is it detecting: Direct sequencing
What is the sample used, and what is it detecting: Whole exome sequencing
Point mutations/SNPs in the entire genome
What is the sample used, and what is it detecting: SNP typing
SNPs in the entire genome
What is the sample used, and what is it detecting: Polymerase chain reaction
Small insertions and deletions
What is the sample used, and what is it detecting: Reverse transcriptase PCR
Expression levels for a small number of genes
What is the sample used, and what is it detecting: Allele specific oligonucleotides
Point mutations, small insertions/deletion
What is the sample used, and what is it detecting: Gene expression array
Expression levels of thousands of genes-not intergenic region or introns
What is the sample used, and what is it detecting: methylated DNA PCR
Epigenetic changes, DNA methylation
What is the sample used, and what is it detecting: comparative genome hybridization
insertions, deletions, and aneuploidies in the kilo to megabase range
What is the sample used, and what is it detecting: FISH
Copy number of a selected chromosome
What is the sample used, and what is it detecting: ELISA
Amounts of protein in a sample