DNA hybridisation: DNA Complementarity and its application

Question 1

What do nucleotides make up?

Answer 1

Make up DNA and RNA

Question 2

Components of a nucleotide?

Answer 2

Nitrogenous base
Pentose sugar
Phosphate group

Question 3

List the purines

Answer 3

Adenine

Guanine

Question 4

List the pyrimadines

Answer 4

Cytosine
Thymine
Uracil

Question 5

What are the 3 bondings in a double helix?

Answer 5

• Sugar phosphates which are linked by phosphodiester bonds
• Base stacking by hydrophobic interactions which excludes water from internal structure
• Van der waal forces are small but contribute to stability

Question 6

What is double stranded DNA formed from?

Answer 6

From 2 antiparallel strands

Question 7

In a double stranded DNA, where are the negatively charged phosphate groups located?

Answer 7

Negatively charged phosphates on the outside of the double stranded DNA

Question 8

When DNA is denatured, what happens to the double stranded molecule?

Answer 8

Conversion of double stranded molecule into a single stranded molecule

Question 9

What is disrupted due to denaturation?

Answer 9

Disruption of Hydrogen bonds within double helix

Question 10

What can also induce the disruption of hydrogen bonds?

Answer 10

Induced by strong alkali or urea

Question 11

How can denaturation be measured?

Answer 11

Can be measured optically by absorbance at 260nm

Question 12

What increases due to denaturation and what is it called?

Answer 12

Absorption increases and this is called hyperchromicity

Question 13

What is Tm?

Answer 13

Point at which 50% of all strands separate

Question 14

What is Tm largely dependent on?

Answer 14

Largely dependent on hydrogen bonds:

• GC content
• Length of DNA molecule
• Salt concentration
• pH
• Mismatches

Question 15

What does a higher GC content mean?

Answer 15

Means more H bonds therefore higher Tm

Question 16

Equation for %GC

Answer 16

((G+C)/(G+C+A+T))*100

Question 17

What happens to Tm value due to a larger duplex and why?

Answer 17

The larger the duplex, the higher the Tm

-There’re more H bonds within the molecule therefore greater stability

Question 18

When is there no contribution to Tm due to length of DNA molecule?

Answer 18

Beyond 300bp, little or no contribution to Tm

Question 19

What does salt stabilise?

Answer 19

Salt stabilises DNA duplexes

Question 20

How does an increase in [Na+] contribute to Tm?

Answer 20

Results in a high Tm

-As it overcomes destabilising effect of mismatched base pairing

Question 21

What does an alkali pH cause to DNA?

Answer 21

Alkali resorts in destablisation

Question 22

How does an alkali pH cause denaturation?

Answer 22

The OH- ions disrupt hydrogen bond pairing

Question 23

The effect of alkali pH on Tm

Answer 23

Fewer H bonds therefore lower Tm

Question 24

What is mismatch defined as?

Answer 24

Defined as base pair which is unable to form hydrogen bonds

Question 25

What is renaturation?

Answer 25

Reversal of denaturation

Question 26

What does the formation of structures in renaturation favour?

Answer 26

Formation of structures favours energy minimisation driven by change in free energy

Question 27

What is renaturation facilitated by?

Answer 27

- Slow cooling | - Neutralisation

Question 28

What is hybridization?

Answer 28

Formation of duplex structure of 2 DNA molecules that have been introduced to one another

Question 29

What is thermodynamically favoured over mismatches?

Answer 29

Perfect matches have a higher Tm therefore favoured more than mismatches

Question 30

What allows us to manipulate specificity?

Answer 30

Limiting hybridisation between imperfectly matched sequence

Question 31

What is stable under high stringency and what is it determined by?

Answer 31

Only complementary sequences are stable and is determined by a: - Temperature near Tm - Low salt concentration

Question 32

What does nucleic acid hybridisation technique identify?

Answer 32

Identifies presence of nucleic acid containing specific sequence of bases

Question 33

What does nucleic acid hybridisation allow?

Answer 33

Allows the absolute or relative quantitation of these sequence in a mixture

Question 34

What does hybridisation use to form specific duplexes?

Answer 34

Hybridisation uses the ability of nucleic acids to form specific duplexes

Question 35

What do probes detect?

Answer 35

Probes detect the presence of complementary nucleic acid by hybridisation

Question 36

What molecules are probes?

Answer 36

ssDNA or RNA molecules

Question 37

Length of probes

Answer 37

20-1000 bases in length

Question 38

What are probes labelled with?

Answer 38

Labelled with a fluorescent or luminescent molecules

Question 39

What does NA blotting technique analyse?

Answer 39

Analyses mRNA or DNA

Question 40

Why is nucleic acid blotting technique limited?

Answer 40

Limited as they only detect one gene at a time

Question 41

What is nucleic acid blotting technique superseded by?

Answer 41

Superseded by PCR

Question 42

What are micro-arrays?

Answer 42

Ordered assembly of thousands of nucleic acid probes

Question 43

What are micro-arrays fixed to and what do we do to the sample of interest?

Answer 43

Fixed to solid surface then sample of interest is hybridised to the probes

Question 44

What do micro-arrays detect and what are they used in?

Answer 44

Detect SNP's and used in genome wide association studies