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

Lesson 4 - Denaturation and Renaturation of DNA Flashcards

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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
Q

how to monitor denaturation/renaturation of DNA

A
  • measure absorbance at 260 nm
  • A260 increases as DNA is denatured
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26
Q

A260 increases as DNA is denatured

A

hyperchromic shift

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27
Q
  • 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
A

DNA breathing

28
Q

significance of DNA breathing

A

enable specialized proteins to interact and “read” its encoded information

29
Q

how to denature DNA

A
  1. heat
  2. alkali
  3. melting proteins
30
Q

pH level of alkali that denatures DNA

A

pH > 11.3

31
Q

what does high pH do

A

extensive deprotonation

32
Q

pH of acid that denatures DNA

A

pH < 2.3

33
Q

what does low pH do

A

extensive protonation

34
Q

what denatures DNA at room temperature

A

pure water

35
Q

example of melting proteins

A
  1. RNA polymerase
  2. HDP (helix destabilizing protein) / single-stranded binding protein
  3. helicase
36
Q

ability of the two separated complemetary strands to reform into the ordered state

A

renaturation

37
Q

two requirements of renaturation

A
  1. salt concentration (0.15-0.50 M NaCl)
  2. temperature (20-25C)
38
Q

what does the salt concentration do to help renaturation

A

eliminate electrostatic repulsion between phosphates

39
Q

what does the optimal temperature do to help renaturation

A

disrupt random intrastrand hydrogen bonds

40
Q

two steps in renaturation

A
  1. nucleation
  2. zippering
41
Q
  • contact is made between two complementary regions of DNA
  • slow process
A

nucleation

42
Q
  • connecting of adjoining base residues into a duplex structure
  • faster process
A

zippering

43
Q

how to monitor renaturation

A

hyperchromicity increases / A260 decreases

44
Q

Cot:
Co

A

initial ssDNA concentration (in molecules/Liter)

45
Q

Cot:
t

A

time for renaturation to be completed (in sec)

46
Q

what does the Cot plot show

A

genome complexity

47
Q

What can be determined from the analysis of Cot plot

A
  1. genome size
  2. genome complexity
48
Q

relative proportions of single-copy and repetitive sequences

A

genome complexity

49
Q

DNA sequence complexity shown in Cot plot

A
  1. highly repetitive
  2. middle repetitive
  3. single copy
50
Q

classes of eukaryotic DNA based on sequence complexity

A
  1. unique sequence
  2. moderately repetitive DNA
  3. highly repetitive DNA
51
Q
  • 1 to 3 copies per genome
  • signle copy, low copy, nonrep. DNA
  • most coding genes
A

unique sequence

52
Q

number of unique sequence copies per genome

A

1-3 copies

53
Q

different types of copies in the unique sequence

A
  • single copy
  • low copy
  • nonrep. DNA
54
Q

example of unique sequence

A
  • most coding genes
  • regulatory sequences
55
Q
  • 10 to 10,000 copies per genome
  • generally dispersed repeates
  • occasionally clustered
A

moderately repetitive DNA

56
Q

number of moderately repetitive DNA copies per genome

A

10 - 10,000 copies

57
Q

where are moderately repetitive DNA found

A

generally dispersed

58
Q

example of moderately repetitive DNA

A
  1. rRNA genes
  2. tRNA genes
  3. histone genes
59
Q
  • 100,000 to 1M copies per genome
  • each copy can range from few nts to several hundreds
  • generally found as tandem repeats
A

highly repetitive DNA

60
Q

number of highly repetitive DNA copies per genome

A

100,000 - 1M copies

61
Q

how are highly repetitive DNA copies generally found

A

as tandem repeats

62
Q

example of highly repetitive DNA

A
  1. centromeric DNA
  2. telomeres DNA
  3. satellite DNA
  4. some transposons
63
Q

how does renaturation help in getting the percent homology between two different organismal DNA

A

amount of renaturation reflects similarity

64
Q
  • ssDNA or RNA fragment of known sequence
  • complemetary to the target DNA/RNA
  • labeled radioactively or non-radioactively
A

probe

65
Q

number of nucleotides in probe

A

15 - 1000 nt

66
Q

how are probes labeled

A
  • radioactively
  • non-radioactively
67
Q

what is the purpose for labeling probes

A

detects specific nucleotide sequence in DNA/RNA sample

BIO107 - Cell and Molecular Biology (8 decks)

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