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Molecular Biology


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Whats different betwn bacterial Phenotype vs. Genotype?
Phenotype = visible properties
Genotype = genetic properties
What is a Probe?
A labeled sequence of ssDNA or ssRNA that hybridizes with its complement by base pairing.
What are the 5 steps involved in constructing a DNA probe?
1. Identification
2. Isolation
3. Reproduction
4. Seperation
5. Labelling
What is "identification"?
Specifying the sequence of bases in an organism's genome that distinguishes it from others.
How is the unique base sequence isolated from a genome?
By using restriction endonucleases to cut it out.
What 2 cut types do REs make?
-Flush end
-Sticky end
How is the unique nucleic acid sequence reproduced (2 steps)?
1. Cloning
2. Detection of cloning host cells
How does cloning work?
1. DNA fragms + cloning vector
Result = Chimeric plasmid w/ antibiotic resistance factor
2. Put chimeric plasmid in host cells (ecoli) on media with antibiotic
3. Host makes lots of copies
4. Check for antibiotic resistance to make sure the plasmid is chimeric.
How is the foreign (probe) dna seperated from the chimeric plasmid?
Cleave w/ RE and then seperate w/ gel electrophoresis.
What are 5 types of labels used for probes?
1. Radioactive
2. Enzymatic
3. Immunologic
4. chemiluminescent
5. Fluorescent
How is a probe used to identify microorganisms?
1. Denature target DNA to ssDNA
2. Add probe; look for hybridization.
What are 3 types of hybridization?
1. Solid support (nitrocellulose filter or plastic well)
2. In solution
3. In situ
How is a nitrocell filter prepared?
By baking the target ssDNA on the filter at 80 C for 2 hrs, then adding the probe.
What is FISH?
Flourescent In Situ Hybridization
What are some advantages of Probes?
1. Fast ID of fastidious pathogens
2. Can ID pathogens unable to grow in culture.
3. Highly specific
4. Can detect non-viable pathogens.
What are some disadvantages of Probes?
1. Expensive
2. Can't detect pathogens you don't suspect.
3. May detect non-viable insignificant pathogens.
4. Still have to grow for antibiotic suscept testing
5. Lack of FDA approval
6. Hard to choose best probe
7. Lack of sensitivity
What is NAAT?
Nucleic acid amplificn tests - a technique used for rapidly amplifying specific unique regions of DNA.
What is a common type of NAAT?
PCR - Polymerase Chain Reaction
What are the 4 steps in PCR?
1. Heat/denaturation of dsDNA
2. Cool/annealing of primers
3. Taq polymerization of new DNA
4. Repeat many times
What is a simplified way of thinking about PCR?
Find a needle in a haystack, then make a haystack of needles.
How is the PCR product analyzed?
By hybridizing with a specific probe.
Why do you use PCR?
To make a ton of copies of a probe, so you get a better signal when identifying microorganisms.
What are 3 advantages of PCR?
1. Can detect a v. small amt of microbial cells
2. Highly sensitive/specific
3. Good for ID pathogens straight from the specimen.
What pathogen is PCR mostly used for detecting currently?
Chlamydia trachomatis.
What are 5 limitations of PCR?
1. Requires expert technical competency
2. Subject to contamination
3. Needs dedicated lab space
4. Sample prep is laborious
5. Expensive for small labs.
How is In Situ PCR done?
Patient sample fixed to slide, hybridization of probe occurs right there in the tissue!
What is Real Time PCR used for?
Detecting and quantifying nucleic acid, AS the replication is occuring.
What type of probes are used in RT-PCR?
FRET - Flourescence Resonence ENergy Transfer probes - have Flourescent/Quencher labels, so when hybridization occurs, the quencher comes off and allows flourescence.
What does RFLP stand for?
Restriction fragment length polymorphism
What are RFLPs used for?
DNA fingerprinting
How are RFLPs made (2 steps)?
1. Cleave chromosomes or plasmids with RE.
2. Seperate DNA fragments with PFGE
what is PFGE?
pulsed field gel electropheresis - a multidimensional current passes through and allows much better seperation.
What are 3 uses for RFLP?
1. Epidemiology
2. Infection control
3. Forensics
What are the 4 steps in a Southern blot procedure?
1. Cleave DNA with RE
2. Electrophorese DNA fragments in agarose gel to seperate out
3. Transfer DNA bands to membrane
4. Detect specific target DNA fragments with a probe.
What is a
Southern = DNA probe for DNA
Northern = DNA probe for RNA
Western = Ab for protein
What is Microarray essentially?
Biochip technology;
1. Chip has nucleic acid array.
2. Patient sample added
3. Detect hybridization
What are 2 methods for detection in microarray?
1. Fluorescence (the sample nucleic aa is labeled(
2. Mass spectrometry

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