DNA Hybridisation: DNA Complementarity, Hybridisation And Its Application

Question 1

Where does the nitrogenous base of DNA join the sugar?

Answer 1

C1

Question 2

Where does the phosphate group of DNA join the sugar?

Answer 2

C5

Question 3

Where does the hydroxyl group of DNA join the sugar?

Answer 3

C3

Question 4

Which carbons in the pentose ring is the oxygen bridge between?

Answer 4

C1-C4

Question 5

Which are the pyrimidine bases?

Answer 5

Cytosine and thymine

Question 6

Which are the purine bases?

Answer 6

Guanine and adenine

Question 7

What is base stacking caused by?

Answer 7

Hydrophobic interactions that change the arrangement of bases set above each other on the inside of the DNA structure

Question 8

How many hydrogen bonds does the C-G base pairing form?

Answer 8

3

Question 9

How many hydrogen bonds does the A-T base pairing form?

Answer 9

2

Question 10

Under what conditions does DNA denature?

Answer 10

When the DNA in solution is heated or exposed to a strong alkali and urea

Question 11

What does the single DNA strand look like?

Answer 11

A randomly coiled string

Question 12

How can you measure the denaturation point of DNA?

Answer 12

Using optical density at 260nm

Question 13

What happens to the absorbance of DNA at higher temperatures?

Answer 13

Increases

Question 14

Why does the absorbance of DNA increase at higher temperatures?

Answer 14

Because single stranded DNA absorbs more

Question 15

What is hyperchromicity?

Answer 15

Increased absorption of light at 260nm on denaturation

Question 16

What is the melting temperature (Tm) of DNA?

Answer 16

The point at which half of the DNA strands have separated

Question 17

What factors determine the melting temperature of DNA?

Answer 17

GC content, length of DNA molecule, salt concentration, pH and mismatches

Question 18

How does GC content determine the melting point of DNA?

Answer 18

Higher GC content (more guanine-cytosine bps) -> more H bonds -> higher melting point

Question 19

How does length of the DNA molecule determine the melting point of DNA?

Answer 19

Longer = higher melting point

Question 20

Why does length of the DNA molecule determine the melting point of DNA?

Answer 20

More H bonds means it’s more stable

Question 21

How does salt concentration determine the melting point of DNA?

Answer 21

Salt stabilises the DNA helix so higher sodium conc increases the melting point

Question 22

What does high salt conc reduce in DNA at a given temperature?

Answer 22

The specificity of base pairing

Question 23

How does pH affect the melting point of DNA?

Answer 23

Alkalinity disrupts the H bonds and lowers the melting point

Question 24

How do mismatches affect the melting point of DNA?

Answer 24

More mismatches mean a lower melting point

Question 25

Why do mismatches affect the melting point of DNA?

Answer 25

Less H bonds, so less stable

Question 26

What's the opposite of denaturation?

Answer 26

Renaturation

Question 27

What is renaturation facilitated by?

Answer 27

Slow cooling and neutralisation

Question 28

What is the basis for specificity of DNA renaturation?

Answer 28

Complementarity

Question 29

What is stringency?

Answer 29

Limiting hybridisation between imperfectly matched sequences

Question 30

What does stringency allow us to manipulate?

Answer 30

Specificity

Question 31

What is high stringency determined by?

Answer 31

A temperature near the melting point or a low salt conc

Question 32

What happens to DNA at high stringency?

Answer 32

Only complementary sequences are stable

Question 33

What techniques do complementarity and hybridisation underlie?

Answer 33

Northern and southern blotting, microarrays, dideoxy (Sanger) and next gen sequencing, PCR and cloning

Question 34

What is northern and southern blotting?

Answer 34

An analysis of mRNA or DNA

Question 35

What limits the use of northern and southern blotting?

Answer 35

It can only detect one gene at a time in small numbers of samples, which makes it slow. Also gel based so time consuming and messy

Question 36

How do microarrays work (basic)?

Answer 36

Thousands of nucleic acid probes fixed to a solid surface, then a sample of interest is hybridised to the probes

Question 37

What do nucleic acid hybridisation techniques allow?

Answer 37

The absolute or relative quantitation of sequences in a mixture

Question 38

Basically, how do nucleic acid techniques work?

Answer 38

Label a probe and hybridise it in a mixed population of DNA. This captures specific DNA sequences

Question 39

How does hybridisation form specific duplexes?

Answer 39

Using the complementarity of nucleic acids

Question 40

What are the characteristics of a probe DNA molecule?

Answer 40

Single stranded, 20-1000 bases in length, labelled with a fluorescent or luminescent molecule

Question 41

What is the process of northern or southern blotting?

Answer 41

Extract DNA or RNA Gel electrophoresis Transfer to nylon membrane Add labelled probe and let it hybridise to the sample Detect hybridisation using electrophoresis