Dna Tech Midterm

Front 1

Nuclease

Back 1

An enzyme that cleaves phosphodiester bonds in nucleus acids

Front 2

DNase

Back 2

A nuclease that cleaves the backbone of DNA molecules, not RNA

Front 3

RNase

Back 3

A nuclease that cleaves the backbone of RNA molecules, not DNA

Front 4

Exo vs. Endonuclease

Back 4

Cleave ends versus center/body of nucleic acid molecules

Front 5

Restriction enzyme

Back 5

Enzyme that recognizes a specific site on nucleic acid molecules and creates a db-stranded break

Front 6

Recognition site

Back 6

DNA/RNA sequence of 4-8bp specifically recognized by restriction enzymes - they are palindromes (opposite strand is the same but backwards, inverted repeats)

Front 7

Modification enzyme

Back 7

Enzymes that bind specific recognition sites in self-DNA and methylate A and C, protecting those sites from degradation by restriction enzymes

Front 8

Type 1 vs. Type 2 restriction enzymes

Back 8

Type 1 cuts far (up to 1,000bp?) away from rec site, type 2 cuts within the recognition site (more useful and predictable for biotech)

Front 9

Blunt vs. Sticky ends

Back 9

Nucleic acid molecules that are cut straight down the middle with no overhanging single-stranded DNA (blunt) or a zig-zagged with overhanging ssDNA (sticky)

Front 10

Isoschizomer

Back 10

restriction enzymes that share the same site, but don't necessarily have the same cut pattern (one could be blunt, the other sticky, etc)

Front 11

Star activity and 5 factors that can cause it

Back 11

altered restriction enzyme specificity, leading to erroneous fragment lengths.

1. high glycerol concentration

2. overly high enzyme concentration

3. wrong buffer

4. organic solvents present (DMSO, ethanol)

5. wrong ions used (we need Mg2+)

Front 12

5 restriction enzyme buffer components and their functions

Back 12

1. TrisCl as pH buffer

2. Mg2+ to activate DNA binding proteins

3. DTT to prevent disulfide bond formation within enzyme

4. NaCl to optimize conditions for enzyme activity

5. BSA to bind to the tube and contaminants, stopping the enzyme from interacting with those

Front 13

RFLPs

Back 13

Restriction length polymorphisms; related DNA molecules that differ just enough (at rec. sites) to have different length fragments when treated with restriction enzymes. Used in forensics.

Front 14

restriction mapping for linear vs. circular DNA molecules

Back 14

linear: 1 cut = 2 fragments

circular: 1 cut = 1 fragment

Front 15

BSL-1: organism properties + examples

Back 15

1) well-characterized 2) rarely disease-causing in healthy people 3) limited risk

ex: E. coli K-12, Staphylococcus epidermidis, Saccharomyeces cerevisiae

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BSL-2: organism properties + examples

Back 16

1) infectious, but not airborne 2) any cell culture 3) bodily fluids

ex: S. aureus, herpes simplex viruses, most flus, Clostridium tetani, salmonella sp.

Front 17

BSL-2+: organism properties + examples

Back 17

Dangerous, incurable, not vaccine-preventable - but not AIRBORNE.

ex. HIV

Front 18

BSL-3: organism properties + examples

Back 18

seriously harmful, often airborne, high severity diseases

ex. Coxiella burnetii, Mycobacterium tuberculosis (TB)

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BSL-4: organism properties + examples

Back 19

dangerous, exotic pathogens w/o cures that tend to be lethal.

ex. Ebola/Marburg viruses

Front 20

Standard precautions

Back 20

hand washing before and after, PPE for eyes and clothing, surface disinfection, biosharps waste management

Front 21

glove disposal area

Back 21

lidded biohazard trash can

Front 22

disposal area of anything with EtBr on it (used buffer, waste gels, pipette tips)

Back 22

beaker labelled for EtBr waste

Front 23

when using a gel box, which color is (-) or (+)? DNA will migrate towards which side and why?

Back 23

black = (-) anode

red = (+) cathode

DNA is (-) charged, so will go towards red.

Front 24

function of EtBr in gel electrophoresis

Back 24

EtBr doesn't fluoresce in water, but it intercalates between DNA base pairs then fluoresces under UV light -> so, it indicates the position of the DNA in the gel.

Front 25

bromophenol blue vs xylene cyanol as loading dyes

Back 25

- bromophenol blue runs like a 300bp DNA fragment (farther), so it's good to visualize gel electrophoresis progress with shorter fragments.

- xylene cyanol runs more like a 3kb fragment, so better for longer fragments.

Front 26

6 factors affecting DNA migration

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1. buffer composition

2. direction of applied electricity

3. voltage strength

4. concentration agarose (higher = less migration)

5. DNA conformation (circular, linear, or supercoiled)

6. intercalating dyes

Front 27

units of % (m/v) solutions and % (v/v) solutions

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% (m/v): grams solute/mL solution

% (v/v) : mL solute/mL solution

Front 28

why did restriction enzymes evolve?

Back 28

bacterial defense mechanism from foreign DNA, make db-stranded breaks in it

Front 29

how are restriction enzymes named?

Back 29

first letter from genus,

next two letters from species name,

capital letter following those from the serotype/strain,

the Roman numerals represent the order of discovery of that enzyme from the same organism

ex. the first enzyme isolated from E. coli strain RY13 was EcoRI.

Front 30

4 components of a restriction digest

Back 30

1. dH2O

2. the buffer - supplied 10X, need 1X

3. DNA - less than 150ng/ul

4. restriction enzyme, some excess used (10U of enzyme used in lab, where 1U digests 1ug DNA/hr)

Front 31

function of the cos-site in lambda DNA

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the overhanging, ssDNA bits on each end of the lambda DNA are complementary and can bind to each other, making circular DNA. (this must be destroyed by heating for 5min at 65-70C)

Front 32

concatemers

Back 32

partly circularized DNA from cos-end complementary binding of each end in lambda DNA