Brainscape's Knowledge GenomeTM

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


See full index

Genomics > DNA Hybridisation > Flashcards

DNA Hybridisation Flashcards

1
Q

What are nucleotides?

A

Nucleotides are monomeric units of the nucleic acid polymers: DNA & RNA. They consist of:

  • Nitrogenous base (ring structure composed of C or N)
  • Pentose sugar
  • Phosphate group
Pentose Sugar:
• 5 carbons that form a cyclical structure, with an oxygen bridge
• Carbons are numbered 1-5
• Nitrogenous base joined to carbon 1
• Phosphate group joined to carbon 5
• Hydroxyl group carbon 3
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What are the 4 DNA nucleotide bases?

A

pYrimidines: cYtosine, thYmine (single Nitrogen containing ring)
pUrines: gUanine and adenine (double Nitrogen containing ring)

IN RNA, Uracil substitutes Thymine and base pairs with Adenine in RNA to form a duplex structure. Uracil is also a pyrimidine.

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

What forms the basis of the Watson and Crick base pairing?

A

Hydrogen bonding forms the basis of the Watson and Crick base pairing. There are:

  • 2 H-bonds between A & T.
  • 3 H-bonds between C & G.

G-C pairing is stronger than A-U pairing.
Watson and Crick’s bonding is the pairing of nucleotide bases within DNA, and it is largely determined by hydrogen bonding between oppositely charged groups.

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

What are the different types of bonds/forces in DNA?

A

The nucleotide chain forms a double helix which can take on different confirmations. The most common of which is the b-form DNA.

  • Sugar phosphate (backbone): linked by phosphodiester linkage.
  • Base Stacking: hydrophobic interactions -> arrangement of bases set above each other internalised to the structure and exlcludes water. Double stranded DNA derives stability by hydrogen bonding.
  • 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
5
Q

What forms the double stranded DNA?

A

The double stranded helix is formed by two anitparallel strands (opposite orientation). The bases are on the inside (stacked bases) and negatively charged Phosphates on the outside.

DNA therefore has an overall negative charge (characteristic used for gel electrophoresis).

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

How can DNA be denatured?

A
  • Conversion of a double stranded molecule to a single stranded molecule.
  • Disruption of Hydrogen bonds within double helix.
  • This is achieved in solution via chemicals (formamide, urea, alkali) or heat.
  • Denaturation forms a random coil (like string in water)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

How can we measure the denaturation of DNA and what is hyperchromicity?

A

DNA denaturation can be measured via optical density at a wavelength of 260nm. As temperature increases, the duplex melts and the optical density increases, this works because single stranded DNA absorbs UV light to a greater extent than double stranded DNA - hyperchromicity. The denaturation of the DNA duplex depends on the stability of the structure determined by its sequence of bases.

Hyperchromicity: Increased absorption of light at 260nm on denaturation.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the Tm?

A

The temperature at which 50% of all strands separate is called the melting temperature (Tm).

The Tm is specific to a duplex with a given sequence, this can be used to control the formation of a short duplex such as a primer or probe.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What are the 5 factors that the Tm depend on?

A

Tm depends largely on hydrogen bonds:

  • GC content
  • Length of DNA molecule.
  • Salt concentration of the solution
  • pH (alkali is a denaturant)
  • Mismatches (unmatched base pairs).
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Tm and GC content

A

Higher GC content = more H bonds = Higher Tm

%GC = (G + C)/(G + C + A+ T) x 100

3 H-bonds in G:C vs 2 H-bonds in A:T

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Tm and Molecule Length

A
  • The longer the contiguous duplex, the higher the Tm
  • More H-bonds in molecule = greater stability
  • However, little/no contribution added beyond 300 base pairs.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Tm and Salt Concentration [Na+]

A
  • Salt stabilises DNA duplexes.
  • High [NA+] = High Tm.
  • Increasing the salt concentration of the structure increase the Tm and thus overcomes the destabilising effect of mismatched base pairing.
  • High salt reduces the specificity of base pairing at a given temperature. This is because of the stabilising effect of salt and its effect on the changing melting temperature of the duplex.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Tm and pH.

A

Chemical denaturants disrupt H-bonds (alkali, formamide, urea).

Fewer H-bonds = Lower Tm.

  • High pH (alkalinity) destabilises DNA duplexes.
  • OH- disrupts H-bond pairing.
  • NaOH –> Na+ + OH-
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Tm and Mismatches

A

Mismatch - a base pair combination that in unable to form H-bonds.

  • Mismatches reduce number of H-bonds formed in a duplex = lower Tm
  • Shorter contiguous stretches of double stranded sequence = lower Tm.
  • Mismatches also distorts the structure and destabilises adjacent base pairing. These factors combine making the formation of a duplex less energetically favourable, reducing the change in free energy in duplex formation.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is renaturation?

A

The reversal of denaturation. The formation of structure favours energy minimisation driven by change in free energy ΔG.

Facilitated by:

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

What is hybridisation?

A

Formation of duplex structure of 2 DNA molecules that have been introduced to one another. (E.g. short synthetic DNA or primer and genomic DNA).

17
Q

Complementarity and Tm

A

Complementarity and Tm is the basis of specificity.

  • Perfect matches have a higher Tm.
  • Are thermodynamically favoured over mismatches.
  • We can use this property to form a complementary molecule with no mis-matches.
18
Q

What is stringency?

A

Stringency: manipulating conditions limiting hybridisation between imperfectly matched sequences allowing us to manipulate specificity.

Under high stringency: only complementary sequences are stable - determined by a temperature near Tm or low salt concentration.

Low stringency can be used as a the kinetics of hybridisation are much faster under in these conditions.

19
Q

What nucleic acid based techniques are Complementarity and Hybridisation vital for?

A
  • Northern blotting
  • Southern blotting
  • Microarrays
  • Dideoxy and Next Gen Sequencing
  • PCR
  • Cloning
20
Q

What are nucleic acid hybridisarition techniques useful for?

A
  • Identifying the presence of Nucleic Acids containing specific sequences of bases.
  • Allows the absolute or relative quantitation of these sequences in a mixture.
21
Q

How do the nucleic acid techniques provide specificity?

A

Under stringent conditions, hybridisation is dependent upon the complementarity of the sequences forming the duplex and this provides the specificity of the technique. Labelled short DNA molecules, oligonucleotides are used to detect unique sequences and are part of a gene are referred to as a probes.

22
Q

What is a probe?

A

A single stranded molecule used to search for its complementary sequence in a sample genome.

  • A ssDNA (or RNA) molecule
  • Typically 20-1000 bases in length.
  • Labelled with a fluorescent or luminescent molecule (less commonly with a radioactive isotope)
  • In some techniques thousands or millions of probes are used simultaneously (e.g. microarray).
23
Q

What is northern blotting?

A

Northern blotting is an adaptation of southern blotting for analysing genes expressed in cells, tissues or organs.

  • Analysis of mRNA and DNA.
  • Limited technique only detects one gene at a time and small numbers of samples (supserseeded by other techniques such as quantitative/digital PCR, microarrays, NGS).
  • The gel based techniques are time consuming and messy.
  • Largely suspended by quantitative PCR or other techniques.
24
Q

How does the original technique of northern/southern blotting work?

A

Extract DNA/RNA –> separate strands via gel electrophoresis –> transfer via mass flow through capillary action of buffer (carries nucleic acid passively) to nylon membrane where it is covalently bonded to the membrane and then hybridised with probes –> detect hybridisation.

Typically today this is done by electroblotting where the gel is sandwiched between a positive and negative electrode. The voltage applied across the gel electrophoretically transfers the negatively charged nucleic acid to membrane (nylon) and the probes are added (hyridisation) and can be visualised fluorescently.

25
Q

How and why do we use microarrays?

A

Used to analyse the expression of thousands of transcripts in each sample.

Microarrays:

  • An ordered assembly of of thousands of nucleic acid probes.
  • Probes are covalently fixed to a solid surface (silicone beads), then sample of interest is hybridised to the probes.
  • Simultaneously measuring 50,000 different transcripts in a cell, tissue or organ.
26
Q

How can we compare gene expressions?

A

Microarrays can be used to compare gene expressions (e.g. between drug treated cellular RNA labelled and hybridised and untreated cells RNA labelled and hybridised).

The amount and location of the label measured can be compared and we can determine the changes in specific genes and identify signatures that relate to specific disease conditions. This tells us how much of each and every transcript in the human genome is being expressed and is an alternative to RNA-seq (NGS).

27
Q

How can we assess millions of Single Nucleotide Polymorphisms (SNPs)?

A

Microarrays can also asses millions of SNPs.

  • DNA from 1 person on 1 microarray.
  • Detects 2.5 million SNPs simultaneously/
  • Result: homozygous or heterozygous for each SNP
  • Used in Genome Wide Association studies (GWAS).

Cheaper and more common than NGS.

Genomics (11 decks)

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