DNA hybridisation and DNA complementarity

Question 1

What is DNA comprised of?

Answer 1

• 4 nucleotides
• Phosphate
• Hydroxyl group
• Pentose sugar
• Phosphate group

Question 2

What is the nitrogenous base?

Answer 2

• A ring structure composed of carbon and nitrogen.

- Can be double or single ringed.

Question 3

What is the pentose sugar?

Answer 3

-5 carbons that form a cyclical structure with oxygen bridge between C1 and C4 carbons.

Question 4

Which carbon is the nitrogenous base attached to?

Answer 4

-Carbon 1

Question 5

Which carbon is phosphate group attached to?

Answer 5

-Carbon 5 via ester bond

Question 6

Which carbon is hydroxyl group attached to?

Answer 6

-Carbon 3

Question 7

How many hydroxyl groups in RNA?

Answer 7

2

Question 8

What is hydroxyl groups important for?

Answer 8

-Key to polymerisation of nucleotides in DNA or RNA

Question 9

What are pyrimidines?

Answer 9

-Cytosine and Thymine

Question 10

What are purines?

Answer 10

-Thymine and Adenine

Question 11

On what basis does hydrogen bonds form?

Answer 11

-On the basis of Watson Crick pairing

Question 12

How are sugar phosphates linked?

Answer 12

-linked by phosphodiester bonds between 3 prime and 5 prime.

Question 13

How does DNA gain its stability?

Answer 13

• Sugar phosphates
• Base stacking
• VDW
• Hydrogen bonds

Question 14

What is the double stranded helix formed from?

Answer 14

-Two antiparallel strands

Question 15

How are the DNA strands arranged?

Answer 15

• Bases stacked

- Negative phosphates on outside

Question 16

What is denaturation?

Answer 16

• When double stranded DNA becomes single stranded

- Due to disruption of Hydrogen bonds

Question 17

Under what circumstances does denaturation occur?

Answer 17

• Occurs when DNA in solution is heated

- Can also be induced by strong alkali or urea

Question 18

What is hyperchromicity?

Answer 18

-Increased absorption of light at 260nm On denaturation

Question 19

What is Tm?

Answer 19

-Point at which 50% of all strands separate is called the melting temperature or Tm.

Question 20

How can we measure denaturation?

Answer 20

-Denaturation can be measured optically by absorbance at 260nm

Question 21

How does hypochromicity?

Answer 21

• As temperature increases the duplex melts and the optical density increases
• Single stranded DNA absorbs UV light to a greater extent than double stranded DNA, this property is termed hyperchromicity
• The denaturation of a DNA duplex depends upon the stability of the structure determined by its sequence of bases

Question 22

What does Tm depend on?

Answer 22

• GC content
• Length of DNA molecule
• Salt concentration
• pH (alkali is a denaturant)
• Mismatches (unmatched base pairs)

Question 23

What is the result of higher GC content?

Answer 23

• Hydrogen bonds

- Higher Tm

Question 24

What is formula to find percent of GC?

Answer 24

%GC = ((𝐺+𝐶))/((𝐺+𝐶+𝐴+𝑇)) x 100

-Where G is the number of guanine nucleotides, C is the number of cytosine nucleotides etc

Question 25

How does length of molecule affect Tm?

Answer 25

- Longer the contiguous duplex, the higher Tm - More Hydrogen bonds within the molecule greater stability - However little further contribution beyond 300 bp

Question 26

What is the relationship between Na+ and Tm?

Answer 26

-Increasing the salt concentration stabilises the structure increases the Tm and thus overcomes the destabilising effect of mismatched base pairing

Question 27

Which chemical denaturants disrupt hydrogen bonds?

Answer 27

-Alkali, formamide, urea

Question 28

How does NaOH disrupt hydrogen bonds?

Answer 28

- OH- disrupts H bond pairing - Fewer hydrogen bonds = Lower Tm - High pH (alkalinity) destabilises DNA duplexes

Question 29

What is a mismatch?

Answer 29

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

Question 30

What are the effects of mismatches?

Answer 30

- Reduces Number of Hydrogen bonds, Fewer = lower Tm - Shorter contiguous stretches of double stranded sequence = lower Tm - Mismatches also distorts the structure and destabilises adjacent base pairing

Question 31

What is the reversal of denaturation?

Answer 31

-Formation of structure favours energy minimisation driven by change in free energy DG

Question 32

What is renaturation facilitated by?

Answer 32

- Slow Cooling | - Neutralisation

Question 33

Define hybridisation

Answer 33

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

Question 34

At what temperature do we get perfect hybrid?

Answer 34

-85 degrees

Question 35

What is the relationship between perfect matches and Tm?

Answer 35

- Perfect matches have a higher Tm | - Thermodynamically favoured over Mismatches

Question 36

What are the effects of manipulating conditions?

Answer 36

-Limiting hybridisation between imperfectly matched sequences allows us to manipulate specificity

Question 37

What is the effect of high stringency?

Answer 37

-Only a perfectly matched duplex can form

Question 38

What is the effect of low stridency?

Answer 38

-Hybridisation occurs with mismatches allowed.

Question 39

What are the conditions of high stridency?

Answer 39

- Temp near Tm | - Low Na concentration