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ZZZ - BIO107 - Cell and Molecular Biology > Lesson 4 - Denaturation and Renaturation of DNA > Flashcards

Lesson 4 - Denaturation and Renaturation of DNA Flashcards

(67 cards)

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1
Q

process of strand separation

A

denaturation

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2
Q

plot of temperature vs extent of denaturation

A

melting curve

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3
Q
  • midpoint of the temperature range over which the strands of DNA separate
  • rise of A260 is half complete
A

melting temperature (Tm)

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4
Q
  • original strand
  • double stranded
A

native DNA

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5
Q

single stranded DNA

A

denatured DNA

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6
Q

decrease in the absorbance of ultraviolet light in a double stranded DNA compared to its single stranded counterpart

A

Hypochromic

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7
Q

absorbance of double stranded DNA

A

A260 = 1.00

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8
Q

absorbance of single stranded DNA

A

A260 = 1.37

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9
Q

absorbance of bases DNA

A

A260 = 1.60

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10
Q

effect of high percentage of GC content on Tm

A

Tm increases

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11
Q

why does Tm increase when there is high GC content

A

has more hydrogen bonds than AT

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12
Q

effect of urea and formamide on Tm

A

Tm decreases

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13
Q

why does Tm decrease when there is urea and formamide

A
  • capable of hydrogen-bonding with DNA bases
  • maintain unpaired state at temperature where it might normally pair again
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14
Q

effect of methanol on Tm

A

Tm decreases

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15
Q

why does Tm decrease when there is ethanol

A
  • weakens hydrophobic interactions
  • increases solubility of bases
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16
Q

effect of trifluoroacetate on Tm

A

Tm initially rises but then decreases

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17
Q

why does Tm decrease when there is trifluoroacetate

A

weakens hydrophobic interaction

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18
Q

effect of increasing sodium chloride concentration on Tm

A

Tm increases

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19
Q

why does Tm increase when there is an increase in sodium chloride concentration

A

high ionic strength
- neutralize negatively charged phosphates
- stabilize hydrogen bond

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20
Q

effect of formaldehyde on Tm

A

Tm decreases

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21
Q

why does Tm decrease when there is formaldehyde

A
  • it reacts with NH2 group of bases and eliminate their ability to hydrogen-bond
  • cause a slow and irreversible denaturation of DNA
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22
Q

what increases Tm of DNA

A
  1. high GC content
  2. increase sodium chloride (NaCl) concentration
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23
Q

what decreases Tm of DNA

A
  1. urea
  2. formamide
  3. methanol
  4. trifluoroacetate
  5. formaldehyde
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24
Q

melting temperature of DNA

A

70-95C

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25
how to monitor denaturation/renaturation of DNA
- measure absorbance at 260 nm - A260 increases as DNA is denatured
26
A260 increases as DNA is denatured
hyperchromic shift
27
- transient denaturation - small fraction of base pairs in the genome - occurs more frequently between bases with fewer H bonds - necessary for gene regulation - requires on molecules for event to occur
DNA breathing
28
significance of DNA breathing
enable specialized proteins to interact and "read" its encoded information
29
how to denature DNA
1. heat 2. alkali 3. melting proteins
30
pH level of alkali that denatures DNA
pH > 11.3
31
what does high pH do
extensive deprotonation
32
pH of acid that denatures DNA
pH < 2.3
33
what does low pH do
extensive protonation
34
what denatures DNA at room temperature
pure water
35
example of melting proteins
1. RNA polymerase 2. HDP (helix destabilizing protein) / single-stranded binding protein 3. helicase
36
ability of the two separated complemetary strands to reform into the ordered state
renaturation
37
two requirements of renaturation
1. salt concentration (0.15-0.50 M NaCl) 2. temperature (20-25C)
38
what does the salt concentration do to help renaturation
eliminate electrostatic repulsion between phosphates
39
what does the optimal temperature do to help renaturation
disrupt random intrastrand hydrogen bonds
40
two steps in renaturation
1. nucleation 2. zippering
41
- contact is made between two complementary regions of DNA - slow process
nucleation
42
- connecting of adjoining base residues into a duplex structure - faster process
zippering
43
how to monitor renaturation
hyperchromicity increases / A260 decreases
44
Cot: Co
initial ssDNA concentration (in molecules/Liter)
45
Cot: t
time for renaturation to be completed (in sec)
46
what does the Cot plot show
genome complexity
47
What can be determined from the analysis of Cot plot
1. genome size 2. genome complexity
48
relative proportions of single-copy and repetitive sequences
genome complexity
49
DNA sequence complexity shown in Cot plot
1. highly repetitive 2. middle repetitive 3. single copy
50
classes of eukaryotic DNA based on sequence complexity
1. unique sequence 2. moderately repetitive DNA 3. highly repetitive DNA
51
- 1 to 3 copies per genome - signle copy, low copy, nonrep. DNA - most coding genes
unique sequence
52
number of unique sequence copies per genome
1-3 copies
53
different types of copies in the unique sequence
- single copy - low copy - nonrep. DNA
54
example of unique sequence
- most coding genes - regulatory sequences
55
- 10 to 10,000 copies per genome - generally dispersed repeates - occasionally clustered
moderately repetitive DNA
56
number of moderately repetitive DNA copies per genome
10 - 10,000 copies
57
where are moderately repetitive DNA found
generally dispersed
58
example of moderately repetitive DNA
1. rRNA genes 2. tRNA genes 3. histone genes
59
- 100,000 to 1M copies per genome - each copy can range from few nts to several hundreds - generally found as tandem repeats
highly repetitive DNA
60
number of highly repetitive DNA copies per genome
100,000 - 1M copies
61
how are highly repetitive DNA copies generally found
as tandem repeats
62
example of highly repetitive DNA
1. centromeric DNA 2. telomeres DNA 3. satellite DNA 4. some transposons
63
how does renaturation help in getting the percent homology between two different organismal DNA
amount of renaturation reflects similarity
64
- ssDNA or RNA fragment of known sequence - complemetary to the target DNA/RNA - labeled radioactively or non-radioactively
probe
65
number of nucleotides in probe
15 - 1000 nt
66
how are probes labeled
- radioactively - non-radioactively
67
what is the purpose for labeling probes
detects specific nucleotide sequence in DNA/RNA sample

ZZZ - BIO107 - Cell and Molecular Biology (21 decks)

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