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Genomics Yr2 > DNA complementarity hybridisation & application > Flashcards

DNA complementarity hybridisation & application Flashcards

1
Q

What is the basic structure of DNA?

A

Phosphate, nitrogenous base and pentose sugar (Deoxy ribose)

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2
Q

What is the basic structure of RNA?

A

Phosphate, nitrogenous base and pentose sugar (ribose)

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3
Q

What is the structure of the nitrogenous base?

A

Either a single or double ring of nitrogen with polaroid charged groups that result in complimentary base pairing

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4
Q

What is the Structure of the Pentose sugar?

A
  • 5 carbon that form a cyclical structure with oxygen bridge
  • Nitrogenous base bind to carbon 1
  • Phosphate group binds to carbon 5
  • Hydroxyl group at carbon 3
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5
Q

What is the polymer and monomer of DNA?

A

Polymer: Nucleic acid
Monomer: Nucleotides

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6
Q

What are the four nucleotides of DNA?

A

Cytosine, Thymine, Guanine, Adenine

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7
Q

What are the four nucleotides of RNA?

A

Cytosine, Uracil, Guanine, Adenine

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8
Q

Which nucleotides are Purines?

A

Adenine and Guanine.

-single nitrogen ring means they form 2 hydrogen bonds

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9
Q

Which nucleotides are Pyrimidines?

A

Cytosine, Thymine, Uracil.

Double nitrogen ring means they for 3 hydrogen bonds

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10
Q

What determines the specificity of pairing

A

Charged polar groups

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11
Q

What does each complimentary pair contain?

A

Each pair contains a purine and a pyrimidine

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12
Q

How many hydrogen bonds does each base pair have?

A

Cytosine and Guanine form 3 hydrogen bonds

Thymine and Adenine form 2 hydrogen bonds

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13
Q

Which base pairing is stronger?

A

Guanine and Cytosine are stronger as they form a larger number of hydrogen bonds

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14
Q

What bonds are present in a DNA chain?

A
  • Sugar phosphates linked by phosphodiester bonds
  • Base stacking: Hydrophobic interactions from the arrangement of the bases means there is internalized strength that excludes water
  • Van der Waal forces are individually small but contribute to overall stability
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15
Q

What determines the stability of DNA?

A
  • The free energy of the molecule.

- Energy minimization makes double stand DNA dynamic, but can still be influenced by its molecular environment

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16
Q

What are the 3 forms of DNA?

A

A, B,Z

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17
Q

What is the most common form of DNA?

A

B

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18
Q

What provide stability to DNA?

A

Hydrogen bonds, and base stacking

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19
Q

What allows for bases to be stacked?

A

Antiparallel strands

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20
Q

What charge is DNA?

A

Negative due to the phosphate groups, which is useful for gel electrophoresis

21
Q

What can cause for DNA to be denatured?

A

Heat+ Chemicals (Urea, or strong alkali)

22
Q

What is Tm?

A

Melting temperature of which 50% of the strands are denatured

23
Q

What is Hyperchromicity?

A

As the temperature increases the absorbance increases as there is more single stranded DNA which absorbs more UV light then dDNA

24
Q

How does heat and chemicals denature DNA?

A

Disrupt the hydrogen bonds

25
Q

What does denaturation depend on?

A

the stability of structure determined by sequence of bases

26
Q

What does the Tm value depend on?

A
  • GC content
  • Length of DNA molecule
  • Salt concentration
  • pH (alkali is a denaturant)
  • mismatches (unmatched base pairs)
27
Q

How does GC content affect Tm?

A

More GC bonds, more hydrogen bonds, higher Tm

28
Q

Formula for GC content

A

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

29
Q

How does Molecule length affect Tm?

A

More hydrogen bonds, more stability greater Tm, until beyond 300bp were Tm value plateaus

30
Q

How does Salt concentration affect Tm value?

A

Increase [Na] means increase in stability of the structure and a greater Tm

31
Q

How does salt increase stability?

A

Salt reduces the specificity of base pairing and go negates the effect of mismatch base pairing thus increasing Tm

32
Q

How does pH affect Tm?

A

High pH destabilises the duplex so lowers the Tm

33
Q

How does alkali affect stability?

A

Oh ions disrupt H bond and thus reduces stability

34
Q

What is a mismatch?

A

A base pair combination that is unable to form hydrogen bonds

35
Q

How does mismatch affect Tm?

A

Reduces number of Hydrogen bonds, distorts the structure and adjacent base pairing, thus reducing Tm

36
Q

What is renaturation?

A

The reverse of denaturation, formation of the structure favours energy minimization favoured by free energy

37
Q

What facilitated renaturation?

A

Slow cooling and neutralisation

38
Q

What is Hybridisation?

A

Formation of a duplex structure of two DNA molecules that have been introduced to one another

39
Q

What is the basis of specificity?

A
  • Complementarity and Tm.
  • Perfect matches have a higher Tm and are more thermodynamically stable (This property can be exploited to form complimentary molecules with no mis-matches.
40
Q

what is stringency?

A

The manipulation of condition that limit hybridisation between imperfectly matched sequences

41
Q

What conditions are present under high stringency?

A

Temperature near Tm and low salt concentration

42
Q

What nucleic acid based techniques rely on specificity and complementarity?

A
  • Northern blotting
  • Southern blotting
  • Microarrays
  • Dideoxy and Next generation sequencing
  • PCR
  • Cloning
43
Q

What is the main aim of hybridisation techniques?

A
  • Identification of the presence of a Nucleic acid containing a specific sequence
  • Allows the absolute or relative quantitation of these sequences in a mixture
44
Q

How do we achieve the main aim of the hybridisation techniques?

A

using a probe/ primer and allowing for hybridisation to occur between the desired sequence (can be used in diagnostics)

45
Q

What is a probe?

A
  • A ssDNA or RNA

- Labelled with fluorescent or luminescent molecules

46
Q

What is Northern blotting?

A
  • An adaptation of southern Blotting
  • Analysis of mRNA or DNA from a cell, tissue or organ
  • Separation of a gene by gel electrophoresis and using a probe to detect for any hybridisation
47
Q

What is the problem with Northern and Southern Blotting?

A
  • Limited technique as it only detects one gene at a time and small number of samples
  • Time consuming and messy
  • Largely superseded by PCR or other techniques
48
Q

What are microarrays?

A
  • An ordered assembly of thousands of nucleic acid probes
  • Probes are fixed to the surface then sample of interest is hybridised to it
  • Any hybridisation of the probes identifies the molecules present
  • Simultaneously measuring 50,000 different transcripts from a cell, tissue or organ
49
Q

What are the uses of microarrays?

A
  • Compare gene expressions eg during drug trials (determines the changes in specific gene)
  • Help identify genes that relate to a specific disease
  • Detect SNPs (used in GWAS)

Genomics Yr2 (6 decks)

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