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BIOC221 > 9. lab content > Flashcards

9. lab content Flashcards

(39 cards)

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1
Q

steps of molecular cloning

A
  • isolate DNA from any species
  • insert DNA into a cloning vector (recombination)
  • insert the recombinant vector into host cell
  • use host cell to make many copies
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2
Q

molecular cloning

A

creating many copies of a DNA fragment

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3
Q

DNA library

A

A collection of DNA fragments cloned into vectors
useful for
- id of clone fragments
- study of gene structure and function

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4
Q

endo vs exo nuclease

A

endonuclease - breaks phosphodiester bonds of DNA
exonuclease - excises the DNA

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5
Q

restriction enzymes

A

found naturally in bacteria to defend against invading pathogens

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6
Q

cloning vector features

A
  • origin of replication
  • Multiple cloning site
  • colour selection gene
  • selectable marker
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7
Q

expression vector features

A
  • origin of replication
  • selectable marker
  • multiple cloning site
  • colour selection gene
  • strong transcriptional promoter sequence and terminator sequence
  • strong translational initiation sequence and stop codon
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8
Q

how is cystic fibrosis diagnosed

A
  • birth with a heel-prick blood-test
  • if positive further genome sequencing steps taken
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9
Q

DNA level of molecular biology disease research and treatment

A
  • identify variants (disease causing or carriers)
  • gene therapy (eg. CRISPR)
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10
Q

RNA level of molecular biology disease research and treatment

A

Drug targets

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11
Q

Protein level of molecular biology disease research and treatment

A

drug targets

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12
Q

Protein function level of molecular biology disease research and treatment

A

Replacement (eg. insulin for diabetes)

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13
Q

cellular level of molecular biology disease research and treatment

A

cell transplants (eg. insulin producing cells)

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14
Q

System/organ level of molecular biology disease research and treatment

A

Transplant

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15
Q

interpersonal and societal level of molecular biology disease research and treatment

A

genetic counselling

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16
Q

Cystic fibrosis symptoms

Study These Flashcards
A

Lungs
- thick mucus
- predisposed to infection

Intestinal obstructions
Pancreatic disease
Male infertility

17
Q

CF inheritance

Study These Flashcards
A

autosomal recessive

18
Q

common CFTR issues

Study These Flashcards
A
  • splicing effects
  • premature stop codons
  • trafficking defects (F508del)
  • gating defects
  • instability
19
Q

Kalydeco

Study These Flashcards
A

CF treatment
- almost a cure (as long as it’s taken regularly for your entire life)
- only effective for CFTR that gets to cell surface (~8%)

20
Q

Trikafta

Study These Flashcards
A

CF treatment
One potentiator
- helps once CFTR gets to surface
Two Correctors
- correct folding and trafficking

Works for ~90% of CF (have to have 1+ p.F508del gene)
Almost cure

21
Q

Inoculation

Study These Flashcards
A
  • takes cells about 30-60mons to grow and divide and reach exponential growth phase
22
Q

Inducer of lac

Study These Flashcards
A

natural: allolactose (breaks down to glucose and galactose)

lab: IPTG (cannot be metabolised)

23
Q

Beta-galactose reactions catalysed

Study These Flashcards
A

In vivo: lactose -> galactose + glucose

In vitro: ONPG -> galactose + ONP

24
Q

Enzyme unit

Study These Flashcards
A

the amount of enzyme that catalyses the conversion of one micromole of substrate per minute under the specified conditions of the assay method (Wikipedia)

1Eu = 1micromol.min^-1

25
spectrophotmetry
a technique that measures the amount of light absorbed by a solution. The more molecules per volume, the more light absorbed (based on substrate-specific absorbance qualities). There is a linear relationship between absorbance and concentration
26
Standard curve
- the absorbance curve of a standard solution with known concentrations to which test samples can be compared to - allows for quantification of a compound in a test sample - defines the range over which a the Beer-Lambert law applies
27
DNA gel electrophoresis
- separation of linear pieces of DNA based on size - DNA is negatively charged so it moves towards the +ve electrode
28
DNA loading buffer (electrophoresis)
acts as an anchor to prevent the DNA sticking to wells or floating away
29
DNA stain (electrophoresis)
- eg. ethidium bromide or ‘red safe’ Ethidium bromide - binds to DNA and fluoresces when exposed to UV light, allowing visualisation Red safe - binds DNA as well - shorter half-life - can be visualised under blue or white light
30
DNA ladder (electrophoresis)
a set of DNA fragments of known sizes - allows for size estimation of DNA molecules
31
Sheep genome project goals
- improved sheep breeding - increased NZ sheep value - disease management in humans and sheep
32
CFTR protein
- cell membrane chloride-ion channel - regulates water movement in and out of cells - ensures thin, free-moving mucus production
33
mucus
- slippery bodily fluid that lubricates and protects the lining of internal structures
34
sheep as a model for CF
- genetic similarities (CFTR 91% identity, 95% similarity) - anatomical similarities (lungs are comparable size) - functional similarities - physiological similarities
35
identity percentage
percentage of identical amino acids between two sequences
36
similarity percentage
percentage similarity when non-identical but similar amino acids are considered
37
Beer-lambert law
applies to a range over which the relationship between amount/concentration and absorbance is linear - doesn’t apply at high concentrations as molecules interact with each other
38
Diauxic growth
- cells grow in two phases - caused by the presence of two sugar in growth media where one is more readily metabolised by the cell
39
why use two restriction enzymes in cloning?
then the insert can only be inserted in one orientation

BIOC221 (10 decks)

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