DNA complementarity, hybridisation and its effects Flashcards
What 3 structural elements does a nucleotide contain?
- Nitrogenous base
- Pentose sugar
- Phosphate group
What number carbon within the pentose sugar is the nitrogenous base joined to?
Carbon 1
What number carbon within the pentose sugar is the phosphate group joined to?
Carbon 5
What other structure is within the pentose sugar and what number carbon is it joined to?
Hydroxyl group joined to carbon 3
What are the two categories that the nitrogenous bases are classified into? For each category name the 2 bases that belong to it
- Pyrimidines (single ring) - Thymine and cytosine
- Purines (double ring) - Adenine and guanine
Why can a pyrimidine base only hydrogen bond with a purine base?
Because the pairing of a pyrimidine and purine base is energetically favourable as the bases are the right distance apart to allow hydrogen bonding to occur.
Why is it the case that Cytosine (Pyrimidine) can only hydrogen bond with Guanine and not Adenine (which like Guanine is also a purine base)?
Because Cytosine and Guanine are both able to form 3 hydrogen bonds while Adenine is only able to form 2 hydrogen bonds so it has to pair with Thymine which can only form 2 hydrogen bonds as well.
What does the fact that a Cytosine-Guanine pairing can form 3 hydrogen bonds mean for the strength of the pairing when compared to a Adenine-Thymine pairing?
It means that the Cytosine-Guanine pairing is stronger than an Adenine-Thymine pairing as it contains one more hydrogen bond per pair.
Why is the stability of a DNA molecule with more C-G pairings higher than a DNA molecule with less?
More G-C pairings means that for there are more hydrogen bonds within the DNA molecule making it harder to break down (more stable).
Name the different forces/bonds within a polypeptide chain of DNA
- Phosphodiester bonds
- Hydrogen bonds
- Base stacking
- Van der waals forces
Explain the concept of base stacking
The arrangement of the bases stacked one above the other within the DNA helix. There are hydrophobic interactions between the stacked bases which excludes water from the internal structure of the double helix.
Explain the concept of Van der waals forces
Interaction between the nuclei of individual atoms within a molecule. This interaction causes the nuclei to attract each other when they’re too far away and repel each other when they’re too close.
Why is DNA a negatively charged molecule?
Because the negatively charged phosphate groups are on the outside of the double helix
What does it mean to “denature” DNA?
Denaturing DNA means to disrupt the hydrogen bonds within a DNA molecule causing it to be converted from one double stranded molecule into 2 single stranded molecules.
What are the 2 main ways of denaturing a DNA molecule?
- Heat DNA in solution
- Induce denaturation using a strong alkali or urea
How can the denaturation of DNA be measured?
Heat up a solution containing Double stranded DNA molecules and measure the absorbance of UV light at 260nm
When measuring DNA denaturation, does the absorbance of UV light at 260nm increase or decrease? Why is this the case?
Absorbance of UV light increases as the DNA denatures. The reason for this is because as you denature the DNA you produce single stranded DNA molecules and these absorb more UV light than double stranded DNA molecules.
What is the increase in absorbance of UV light as a result of DNA denaturation called?
Hyperchromicity
What is Tm (melting temperature)?
The temperature at which 50% of the DNA strands have been separated during a Denaturation reaction
What are the 5 factors that can affect Tm?
Guanine-Cytosine content
Length of DNA molecule
Salt concentration of the environment
pH of environment (alkali is a denaturant)
No. Mismatches (unmatched base pairs)
How does Guanine-Cytosine content affect Tm?
More guanine-cytosine pairs within a DNA molecule means more hydrogen bonds which means a higher Tm as the DNA is more stable.
How does molecular length affect Tm?
The longer the molecule of DNA the more hydrogen bonds that molecule will have meaning it will be more stable and will therefore have a higher Tm.
However, after 300bp there isn’t any further increase in Tm.
How does salt concentration [Na+] affect Tm?
The positive charges of the [Na+] ions neutralise the negative charges of the phosphates thus reducing the repulsion force between the phosphates. This increases the stability of the DNA molecule and therefore Tm
How does pH affect Tm?
Increase in pH causes a decrease in DNA stability and therefore a decrease in Tm. This is because increased pH means increased [OH-]. The OH- ions are able to penetrate into the internal structure of the DNA and disrupt the hydrogen bonds between the bases leading to denaturation.
