(5) Molecular Diagnostics Flashcards
(45 cards)
What are two techniques used to detect infectious agents and diagnose inherited disorders?
- Hybridization
- PCR
Hybridization:
Describe
Single stranded DNA binds to another strand of DNA or RNA with complementary sequence to form:
DNA-DNA hybrid or DNA-RNA hybrid
Hybridization:
useful for?
Detection and quantification of target DNA or RNA
Hybridization:
What is a probe?
Short, single-stranded oligonucleotide
Hybridization:
What do you do with Target DNA?
Converted to single stranded DNA then IMMOBILIZED on a solid support; called blotting
Hybridization:
What is southern blotting?
Probe=DNA
Target=DNA
Hybridization:
What is northern blotting?
Probe=Single stranded DNA
Target=mRNA
Hybridization:
What do probes have on them?
Labeled with radioactive or fluorescent tag
Blotting Techniques:
Purpose?
Detection and visualization of specific biomolecules
Blotting Techniques:
Southern: Target?
DNA
Blotting Techniques:
Southern: Purpose?
Determine which restriction fragments are associated with a gene
Blotting Techniques:
Northern: Target?
RNA
Blotting Techniques:
Northern: Purpose?
Measure size and quantities of mRNA molecule
Blotting Techniques:
Western: Target?
Protein
Blotting Techniques:
Western: Purpose?
Measures amount of protein or antibody
Blotting Techniques:
Eastern: Target
PTM (lipid, carb, phosphorylation)
Blotting Techniques:
Eastern: Purpose?
Detects post-translational modifications (PTMs) on proteins
Polymerase Chain Reaction (PCR)
Describe
DNA subjected to high temp to denature
Primers complement sequences to flank each end of DNA
Allowed to anneal
Add 4 dNTPs
Taq Polymerase; synthesizes copy of DNA by extending the primers on both ends
Polymerase Chain Reaction (PCR)
Advantage?
Very small amount of template DNA needed!
10^9 fold amplification from trace amount of DNA
Polymerase Chain Reaction (PCR)
Disadvantage?
Need to know the sequence of the flanking DNA for primer design, error prone, amplification of contaminating DNA
Polymerase Chain Reaction (PCR)
What are the 3 major steps?1
- Heat to separate
- Cool to anneal
- DNA synthesis
Quantitative PCR (qPCR)
Used for?
Quantifying copy number of a specific gene in two or more samples in real time
- Detect levels of infectious agent
- Determine levels of gene expression
Quantitative PCR (qPCR)
How does it differ from traditional PCR?
In addition to primers, includes…
[a probe that fluoresces only in presence of the PCR product]
Detections of Variations in DNA sequences:
2 methods?
- Restriction Fragment Length Polymorphism (RFLP)
2. Variable number of tandem repeats (VNTR)
