Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

genomics notes > DNA hybridisation > Flashcards

DNA hybridisation Flashcards

1
Q

What are 3 components of DNA?

A
  1. ribose sugar
  2. phosphate group
  3. nitrogenous base
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

How many rings do purines and pyrimidines have?

A
  • 2 rings = purines

- 1 ring = pyrimidine

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are properties of nucleotide chain of DNA?

A
  1. base stacking : hydrophobic interactions > arrangement of bases set above each other internalised to the structure & excludes water.
  2. sugar phosphates : linked by phosphodiester bonds
  3. Van der waaals forces : individually small but contributes to the stability
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is DNA denaturing?

A
  • conversion of a double stranded molecule -> a single stranded molecule
  • disruption of hydrogen bonds within the double helix.
  • occurs when DNA in solution is heated
  • can also be induced by strong alkali or urea
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

how is denaturation measured?

A
  • optically measured by absorbance at 260nm

- as temp increases duplex melts and optical density increases.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is Tm?

A
  • melting temperature
  • point at which 50% of all strand separate is called Tm
  • Tm = 85 degrees
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What is hyperchromicity?

A
  • hyperchromicity = increased abs of light at 260nm on denaturation
  • single stranded DNA absorbs more UV light > double stranded
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What factors does Tm depend on?

A 
- depends largely on hydrogen bonds
=> GC  content 
=> length
=> salt conc
=> pH (alkali is a denaturant)
=>mismatches
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What is the equation to calculate GC content?

A

% GC = (G +C)/ (G+ C+ A+ T) x100

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

how does Tm and length of molecule relate and effect stability?

A
  • longer = higher
  • more hydrogen bonds within molecule increasing stability
  • plateus after 300 bp
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

how does Tm and salt conc relate and effect stability?

A
  • salt stabilises DNA duplex (Na+ is positive charge and DNA is negative charge due to phosphate)
  • high [Na+] = high Tm
  • overcomes the destabilising effect of mismatch base pairing
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

how does pH relate to Tm and effect stability?

A
  • chemical denaturants disrupt hydrogen bonds eg: alkali , formamide, urea
  • OH- disrupts the H+ bond pairing
  • fewer hydrogen bonds = lower Tm
  • high pH (alkalinity) destabilises DNA duplex
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Define mismatch.

A
  • a base pair combination that is unable to form hydrogen bonds.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

how does mismatch relate to Tm and effect stability?

A
  • reduces number of hydrogen bonds
  • fewer H bonds = lower Tm
  • shorter stretched of double stranded sequence = lower Tm
  • mismatches distort the structure and destabilise adjacent base pairing.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is renaturation?

A
  • denaturation is reversible we call this renaturation

- renaturation is favoured by energy minimisation driven by change in free energy

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What facilitates renaturation?

A
  • slow cooling

- neutralisation

17
Q

What causes specificity pairing?

A
  • complementary and Tm is the basis of specificity
  • higher Tm = perfect match bc perfect matches are thermodynamically favoured over mismatches.
  • we can use this property to form a complementary molecule with no mis- matches.
18
Q

Define stringency

A

manipulating conditions , limiting hybridisation between imperfectly matched sequences allows us to manipulate specificity.

19
Q

What are high stringency conditions?

A
  1. high temp , near Tm
  2. low salt concentration
    => only complementary sequences are stable.
20
Q

What does nucleic acid hybridisation technique do?

A
  • identifies the presence of NA containing a specific sequence of bases
  • allows the absolute or relative quantitation of these sequences in a mixture.
21
Q

What does hybridisation use to form specific duplexes?

A
  • uses complementarity and hybridisation of labelled nucleic acids
  • these molecules are referred to as probes.
22
Q

What are characteristics of a probe?

A
  • a ssDNA (or RNA) molecule
  • typically 20 - 1000 bases in length
  • labelled with fluorescent or luminescent molecule
  • sometimes thousands and millions of probes are used simultaneously
23
Q

What is Nothern blotting?

A
  • used for analysis of mRNA or DNA
    1. extract RNA or DNA
    2. gel electrophoresis
    3. transfer to nylon membrane
    4. add labelled probe which hybridises to mRNA transcript in sample.
    5. Detect hybridisation
24
Q

What are limitations of Northern blotting?

A
  • gel based technique so time consuming
  • limited technique only detects one gene at a time and small numbers of samples.
  • PCR and other techniques preferred over it.
25
Q

What is microarrays?

A
  • An ordered assembly of thousands nucleic acid probes
  • probes fixed to a solid surface , then sample interest is hybridised to the probes
  • simultaneously measuring 50, 000 different transcripts in a cell, tissue or organ.
26
Q

What is the advantage of microarrays over blotting techniques?

A
  • they can simultaneously measure the expression of many different transcripts and compare multiple experimental conditions whereas northern blotting can measure only one gene targets.

genomics notes (18 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions