Brainscape's Knowledge GenomeTM

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


See full index

SGUL - Genomics > DNA and Hybridisation > Flashcards

DNA and Hybridisation Flashcards

1
Q

What type of polymers are DNA and RNA?

A

Polynucleotide Polymers

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

What does the DNA consist of?

A

A Pentose sugar
Nitrogenous base
Phosphate group

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

Describe the Pentose sugar.

A
  • 5 carbons that form a cyclical structure with oxygen bridge
  • Carbons are numbered 1-5
  • Nitrogenous base joined to carbon 1
  • Phosphate group joined to carbon 5
  • Hydroxyl group on carbon 3
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

How many Nucleotides make up the DNA?
Which groups are they divided into?

A

Purines - Adenine, Guanine
Pyramidines - Cytosine, Thymine

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

What do the hydrogen bonds in DNA form the basis of?

A

Watson Crick base pairing rule
- Opposite charge distribution results in formation of dipoles

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

Which nucleotides bind to each other?
How many Hydrogen bonds form between them?

A

Adenine - thymine = 2 Hydrogen bonds
Cytosine - Guanine = 3 Hydrogen bonds

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Describe the nucleotide chain of DNA

A
  • Sugar Phosphates: linked by phosphodiester bonds
  • Base stacking : hydrophobic interactions ->arrangement of bases set above each other internalised to the structure & excludes water
  • Van der Waals forces: Individually small but contributes to the stability
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Describe the double stranded DNA.

A
  • Forms 2 anti-parallel stands
  • Negatively charged phosphates on the outside
  • forms a double helix shape
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How can the DNA be denatured? (Basis)

A

DNA double helix + Chemicals/Heat = Denatured

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

How can the DNA be denatured? (STEPS)

A
  • Conversion of a double stranded molecule → of single stranded molecules
  • 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
11
Q

How can Denaturation be measured?

A
  • It can be measured optically by absorbtion at 260nm through Hyperchromicity.
  • Increased absorption of light at 260nm On denaturation Point at which 50% of all the strands are separate is called the melting temperature or Tm
  • The Tm of a duplex is dependent upon the sequence and composition of its bases
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What does TM largely depend on in terms of hydrogen bonds?

A
  • GC content
  • Length of DNA molecule
  • Salt Concentration
  • pH (alkali is a denaturant)
  • Mismatches (unmatched base pairs)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What is the correlation between TM & GC content? Give the equation

A
  • The higher the GC content = The more hydrogen bonds = The higher the TM
  • %GC = TBC
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How does the Molecule length affect the TM?

A
  • The longer the contiguous duplex, The higher the TM
  • More hydrogen bonds within the molecule will have greater stability
  • However little further contribution beyond 300 bp
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

How does the salt concentration affect the TM?

A
  • Salt stabilises DNA duplexes
  • High [Na+] = High TM
  • Increasing the salt concentration stabilises the DNA which increases TM and thus overcomes the destabilising effect of the mismatched base pairing
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How does the salt concentration affect the TM? (PART 2)

A

High salt reduces the specificity of base pairing at a given temperature

17
Q

How does the pH affect the TM?

A
  • Chemical denaturants disrupt hydrogen bonds
  • Alkali, Formamide, Urea
  • NaOH -> Na+ + OH-
  • OH- disrupts the H bond pairing
  • Fewer hydrogen bonds = Lower TM
18
Q

What is a Mismatched base pairing?

A

A mismatch is defined as a base pair combination that is unable to form hydrogen bonds

19
Q

How does Mismatches affect the TM?

A
  • Reduces the number of hydrogen bonds, Fewer H bonds = lower Tm
  • Shorter contiguous stretches of double stranded sequence = lower Tm
  • Mismatches also distorts the structure and destabilises adjacent base pairing
20
Q

How can denaturation be reversed?

A
  • Duplex formation is an equilibrium
  • Denaturation is reversible, This is called renaturation
  • Formation of structure favours energy minimisation driven by change in free energy DG
21
Q

What is Renaturation facilitated by?

A
  • Slow cooling
  • Neutralisation
22
Q

How is Complementarity and Tm the basis of specificity?

A
  • Perfect matches have a higher Tm
  • Are thermodynamicslly favoured over mismatches
  • We can use this property to form a complementary molecule with no mis-matches
23
Q

Describe Stringency

A
  • Manipulating conditions: Limiting hybridisation between imperfectly matched sequences allows us to manipulate specificity
    (VD)
24
Q

Describe the conditions of Stringency

A
  • Under high stringency: Only complementary sequences are stable determined by:
  • Temperature near Tm
  • Low salt concentration
25
Q

What are the Nucleic acid hybridisation techniques?

A
  • Identifies the presence of Nucleic Acids containing a specific sequence of bases
  • This allows the absolute or relative quantitation of these sequences in a mixture
26
Q

What is the Hybridisation technique?

A
  • Hybridisation uses the ability of Nucleic acids to form specific duplexes
  • Uses the complementarity and hybridisation of labelled nucleic acids
  • These molecules are referred to as “probes”
27
Q

What is a Probe?

A
  • A ssDNA (or RNA) molecule
  • Typically 20 – 1000 bases in length
  • Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope)
  • In some techniques thousands or millions of probes are used simultaneously
28
Q

Describe the hybridisation technique in more detail

A
  • In-situ hybridisation of tissue sections
  • Chromosome painting of chromosome spreads
  • Analysis of mRNA or DNA by PCR/qPCR
  • Sanger/dideoxy sequencing
  • These techniques are not very scalable and only detects few genes at a time and small numbers of samples
29
Q

Describe the Genome wide technique

A
  • Frequently we want to analyse thousands of segments of DNA or RNA in each sample.
  • So hybridisation has been employed in high throughput techniques like microarrays and next generation sequencing
  • Both techniques are used for measuring gene expression or genomic composition
30
Q

Describe the High Throughput techniques

A

Microarrays:
- An ordered assembly of thousands of nucleic acid probes
- Probes are fixed to a solid surface, then sample of interest is hybridised

Next Generation sequencing:
- is the parallel sequencing of millions of molecules captured in a surface by hybridisation

SGUL - Genomics (21 decks)

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