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Genomics > DNA complementarity hybridisation & its application > Flashcards

DNA complementarity hybridisation & its application Flashcards

1
Q

What is DNA?

A

Polymer or polynucleotide comprising of 4 nucleotides joined together

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2
Q

What does the nitrogenous base comprise of?

A

A single or double ring, which contains nitrogen and carbon

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3
Q

What is attached to carbon 5 of the ribose sugar?

A

A phosphate group

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4
Q

Where is the hydroxy group on the ribose sugar?

A

Carbon 3

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5
Q

Where is the nitrogenous base on the ribose sugar

A

carbon 1

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6
Q

What are the 4 nucleotides?

A

Cytosine, Guanine, Thymine, Adenine

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7
Q

What are the 2 molecular structures that the 4 nucleotides fall into?

A

Pyrimidines

Purines

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8
Q

What are the structural differences between pyrimidines and purines?

A

Pyrimidine - a single nitrogen ring

Purine- a double nitrogen ring

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9
Q

What are the nucleotides that are pyrimidines?

A

Cytosine and thymine

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10
Q

What are the nucleotides that are purines?

A

Guanine and adenine

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11
Q

What are the watson and crick base pairs?

A

Adenine ad Thymine

Cytosine and Guanine

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12
Q

How many hydrogen bonds form between the base pairs?

A

Guanine and cytosine form 3 hydrogen bonds

Thymine and Adenine form 2 hydrogen base pairs

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13
Q

Which base pair is stronger?

A

Guanine and cytosine because they have 3 hydrogen bonds

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14
Q

In RNA which base pair is stronger?

A

Guanine and Cytosine is still the stronger base pair.

Adenine and Uracil only have 2 base pairs

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15
Q

What molecular structure is uracil?

A

A pyrimidine like Thymine

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16
Q

What links the sugar phosphate groups?

A

Phosphodiester bonds

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17
Q

What bonds are present within DNA?

A

Phosphodiester
For base stacking - Hydrophobic interactions
Van der Waals forces (which are weak but help with stability)

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18
Q

What is the form of DNA that is most common?

A

B form

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19
Q

What determines the stability of DNA?

A

Free energy of the molecule and energy minimisation

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20
Q

What can influence the stability of DNA

A

The molecular environment

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21
Q

How is double stranded DNA formed?

A

From 2 anti parallel strands that means they have opposite orientation

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22
Q

What overall charge does DNA have?

A

Negative due to the phosphate groups. This fact is used for gel electrophoresis

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23
Q

DNA denatured

A

Double strand molecules to a single strand molecule

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24
Q

What can cause DNA to denature?

A

The disruption of Hydrogen bonds within the double helix

25
Q

What causes the hydrogen bonds within DNA to break?

A

When DNA is heated

Or when DNA is in a strong alkali, formamide or urea

26
Q

What is hyperchromicity?

A

Increased absorption of light at 260 nm on denaturation

27
Q

What is Tm/ melting temperature?

A

Point at which 50% of all strands separate.

28
Q

What is special about single stranded DNA?

A

Hyperchromicity - Single strand DNA absorbs more UV light than double stranded DNA

29
Q

What does Tm depend on?

A 
GC content 
Length of DNA molecule 
Slat conc (molecular environment)
pH (alkali denatures DNA)
Mismatched Base pairs
30
Q

What does a higher GC content mean?

A

More Hydrogen bonds = higher Tm

31
Q

What does more hydrogen bonds mean for stability?

A

There is greater stability?

32
Q

Is there a difference in stability after 300 bp?

A

No, after 300 bp there is no increase in Tm / stability

33
Q

What cation influences stability?

A

All of them but sodium ions is the most common

34
Q

What does a higher [Na+] mean for Tm?

A

A higher Tm, as salt conc increases stabilisation.

Higher Salt conc also over comes how unstable mismatched base pair are

35
Q

What else does a high salt also do?

A

Reduces the specificity of base pairing

36
Q

What does an alkali solution do to DNA?

A

Alkali dissociates into its ions.

The OH- ion disrupts the hydrogen bonds in DNA

37
Q

Higher pH means?

A

A lower Tm

38
Q

What is mismatch?

A

A base pair combination that is unable to form a hydrogen bond

39
Q

Low no of H bonds?

A

Low Tm

40
Q

What else does DNA mismatch do to DNA?

A

Distorts the structure and destabilise adjacent base pairing

41
Q

How do you reverse denaturation?

A

Slow cooling

Neutralisation

42
Q

What is renaturation / hybridisation?

A

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

43
Q

Are there different factors that influence renaturation and hybridisation?

A

No they are both examples of duplex formation

44
Q

What contributes to a high Tm?

A

Perfect base pairs

45
Q

What is stringency?

A

Manipulating conditions: Limiting hybridisation between imperfectly matched sequences allows us to manipulate specificity

46
Q

Why would we want low stringency?

A

Kinetics of hybridisation are much faster under low stringency conditions

47
Q

What determines high stringency?

A

Temperature near the Tm or a low salt conc

48
Q

What are the 6 nucleic acid based techniques?

A
  • Northen blotting
  • Southern blotting
  • Microarrays
  • PCR
  • Cloning
  • Dideoxy and next gen sequencing
49
Q

Specific aspects of Hybridisation techniques?

A

Identifies the presence of NA containing a specific sequence of bases

50
Q

What are uses of probes?

A

Capture specific species of DNA
Amplify segments of DNA
Or to quantify the no of molecules present

51
Q

What is a probe?

A

ssDNA or RNA
About 20 -1000 bases in length
Labelled with a fluorescent or luminescent molecule

52
Q

The limitation of northern blotting

A

One gene at a time and a small sample size
messy and time consuming
PCR is better

53
Q

Northern and southern blotting

A
  • Uses DNA or RNA that has been separated by gel electrophoresis.
  • Transferred to a mass capillary flow of a buffer from a reservoir with nylon membrane
  • Captured by and covalently bonded to the membrane and then hybridised with a labelled probe
54
Q

Microarrays

A

An ordered assembly of thousands nucleic acid probes

-They are fixed to a solid surface, then the sample of interest is hybridised to the probes

55
Q

Where are the probes fixed to?

A

A solid surface like a glass matrix or silicon

56
Q

What are microarrays used for?

A

Gene expression profiling for drug treated cells and untreated cells

57
Q

What are GWAS?

A

Genome Wide Association studies

Microarrays with SNPs are used for GWAS

58
Q

What is needed for microarrays?

A

RNA
Labelled with a fluorescent molecule
hybridised to the array and amount and location of the label measured

Genomics (19 decks)

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