Molecular Biology

Front 1

Whats different betwn bacterial Phenotype vs. Genotype?

Back 1

Phenotype = visible properties
Genotype = genetic properties

Front 2

What is a Probe?

Back 2

A labeled sequence of ssDNA or ssRNA that hybridizes with its complement by base pairing.

Front 3

What are the 5 steps involved in constructing a DNA probe?

Back 3

1. Identification
2. Isolation
3. Reproduction
4. Seperation
5. Labelling

Front 4

What is "identification"?

Back 4

Specifying the sequence of bases in an organism's genome that distinguishes it from others.

Front 5

How is the unique base sequence isolated from a genome?

Back 5

By using restriction endonucleases to cut it out.

Front 6

What 2 cut types do REs make?

Back 6

-Flush end
-Sticky end

Front 7

How is the unique nucleic acid sequence reproduced (2 steps)?

Back 7

1. Cloning
2. Detection of cloning host cells

Front 8

How does cloning work?

Back 8

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.

Front 9

How is the foreign (probe) dna seperated from the chimeric plasmid?

Back 9

Cleave w/ RE and then seperate w/ gel electrophoresis.

Front 10

What are 5 types of labels used for probes?

Back 10

1. Radioactive
2. Enzymatic
3. Immunologic
4. chemiluminescent
5. Fluorescent

Front 11

How is a probe used to identify microorganisms?

Back 11

1. Denature target DNA to ssDNA
2. Add probe; look for hybridization.

Front 12

What are 3 types of hybridization?

Back 12

1. Solid support (nitrocellulose filter or plastic well)
2. In solution
3. In situ

Front 13

How is a nitrocell filter prepared?

Back 13

By baking the target ssDNA on the filter at 80 C for 2 hrs, then adding the probe.

Front 14

What is FISH?

Back 14

Flourescent In Situ Hybridization

Front 15

What are some advantages of Probes?

Back 15

1. Fast ID of fastidious pathogens
2. Can ID pathogens unable to grow in culture.
3. Highly specific
4. Can detect non-viable pathogens.

Front 16

What are some disadvantages of Probes?

Back 16

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

Front 17

What is NAAT?

Back 17

Nucleic acid amplificn tests - a technique used for rapidly amplifying specific unique regions of DNA.

Front 18

What is a common type of NAAT?

Back 18

PCR - Polymerase Chain Reaction

Front 19

What are the 4 steps in PCR?

Back 19

1. Heat/denaturation of dsDNA
2. Cool/annealing of primers
3. Taq polymerization of new DNA
4. Repeat many times

Front 20

What is a simplified way of thinking about PCR?

Back 20

Find a needle in a haystack, then make a haystack of needles.

Front 21

How is the PCR product analyzed?

Back 21

By hybridizing with a specific probe.

Front 22

Why do you use PCR?

Back 22

To make a ton of copies of a probe, so you get a better signal when identifying microorganisms.

Front 23

What are 3 advantages of PCR?

Back 23

1. Can detect a v. small amt of microbial cells
2. Highly sensitive/specific
3. Good for ID pathogens straight from the specimen.

Front 24

What pathogen is PCR mostly used for detecting currently?

Back 24

Chlamydia trachomatis.

Front 25

What are 5 limitations of PCR?

Back 25

1. Requires expert technical competency
2. Subject to contamination
3. Needs dedicated lab space
4. Sample prep is laborious
5. Expensive for small labs.

Front 26

How is In Situ PCR done?

Back 26

Patient sample fixed to slide, hybridization of probe occurs right there in the tissue!

Front 27

What is Real Time PCR used for?

Back 27

Detecting and quantifying nucleic acid, AS the replication is occuring.

Front 28

What type of probes are used in RT-PCR?

Back 28

FRET - Flourescence Resonence ENergy Transfer probes - have Flourescent/Quencher labels, so when hybridization occurs, the quencher comes off and allows flourescence.

Front 29

What does RFLP stand for?

Back 29

Restriction fragment length polymorphism

Front 30

What are RFLPs used for?

Back 30

DNA fingerprinting

Front 31

How are RFLPs made (2 steps)?

Back 31

1. Cleave chromosomes or plasmids with RE.
2. Seperate DNA fragments with PFGE

Front 32

what is PFGE?

Back 32

pulsed field gel electropheresis - a multidimensional current passes through and allows much better seperation.

Front 33

What are 3 uses for RFLP?

Back 33

1. Epidemiology
2. Infection control
3. Forensics

Front 34

What are the 4 steps in a Southern blot procedure?

Back 34

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.

Front 35

What is a
-Southern
-Northern
-Western

Back 35

Southern = DNA probe for DNA
Northern = DNA probe for RNA
Western = Ab for protein

Front 36

What is Microarray essentially?

Back 36

Biochip technology;
1. Chip has nucleic acid array.
2. Patient sample added
3. Detect hybridization

Front 37

What are 2 methods for detection in microarray?

Back 37

1. Fluorescence (the sample nucleic aa is labeled(
2. Mass spectrometry