Molecular Diagnositics Flashcards
What are two techniques to detect infectious agents and diagnose inherited disorders
- Hybridization
- Polymerase chain reaction
- detection is based on a known pathogen sequence
- diagnosis is based on known Muna genome
Explain how hybridization works
*useful for detection and quantification of target DNA or RNA
- meant to create single stranded oligonucleotides called probes
- ss-DNA binds to another strand of DNA or RNA to form DNA-DNA or DNA-RNA hybrid
TARGET DNA is made to ss-DNA then immobilized on a support and is called blotting
Southern, northern, and western blotting
Southern Blotting vs Northern blotting
Southern blotting- both probe and target nucleic acid are DNA
Northern blotting- probe is as-DNA and target is mRNA
What part of northern or southern blotting is labeled with radioactive fluorescent tag
The ss-DNA probe
Describe the step of blotting technique
- Material is separated by gel electrophoresis (smaller moves faster)
- Transfer to membrane so it can be exposed on the blot
- Add tagged probe to reveal bands of interest (hybridization occurs)
- Solid lines represent bands reactive with probe
- Show on visual: only bands reactive with probe are made visible
What is the purpose of a southern blot
To determine which restriction fragments are associated with a gene
What is the purpose of northern blot
To measure size and quantities of mRNA molecules to answer questions about gene expression
What is the purpose of western blot
*targets protein
To measure the amount of protein or antibody present
Describe the purpose and steps of PCR
Purpose- amplify DNA sample to create multiple copies
- Ds-DNA from pathogen is denatured at high temps to create ss-DMA
- Primers added and anneal to DNA
- Add all 4 dNTPS
- Taq polymerase -synthesizes DNA
What are the advantages and disadvantages of PCR
Advantage- very small amount of template DNA is needed
Disadvantage- you MUST know the sequence of the DNA for primer design
PCR is also called ____
Cell-free cloning
*allows early detection of microorganisms and detection of specific gene mutations
What is qPCR? What is it used for?
Quantitative PCR (or real time PCR)
Used to quantify copy number of a specific gene in two or more samples in real time
[detect levels of infectious agent and determine levels of gene expression)
What is added to qPCR that is not in regular PCR
In addition to primers, qPCR includes probes which flouresce in presence of the PCR product
- probes are usually oligo with a fluorescent tag.
- can measure intensity of light to quantify
What are the molecular techniques used to detect variations in DNA sequence for forensics and diagnostics
- RFLP
2. VNTR
How does RFLP work?
-based off the idea that genes differ in the recognitiion sequence for restriction enzymes
- Sample DNA is added to restriction endonucleases
- DNA is put on gel and southern blot is used with DNA probe
- X-ray film shows bands and if bands match then they are related