Intro and PCR

Question 1

Direction of DNA

Answer 1

5’ to 3’

Question 2

Charge on phosphate group at physiological pH

Answer 2

Negative

Question 3

What does a nucleotide consist of?

Answer 3

Base
Sugar
Phosphate group

Question 4

How long is a typical DNA strand?

Answer 4

10 - 10^9 nucleotides long

Question 5

Structure of bases

Answer 5

Draw

Question 6

Handedness of DNA double helix

Answer 6

Right handed

Question 7

Width of DNA double helix (i.e. helix diameter)

Answer 7

2 nm (20 A)

Question 8

Length of 1 helical turn

Answer 8

3.4 nm (10 base pairs)

Question 9

DNA strands are…

Answer 9

…antiparallel

Question 10

Base pairs are…

Answer 10

…perpendicular to the helical axis

Question 11

Hydrogen bonds between bases

Answer 11

Draw

Question 12

A-DNA

Answer 12

Rare type of structural conformation that DNA adopts under dehydrating conditions (< 75 % humidity)
Similar to B-DNA but with a shorter and more compact structure, because there is less water present in the grooves - deep and narrow major groove and wide and shallow minor groove
Wider and flatter than B-DNA [helix diameter 2.6 nm (26 A)]
Base pairs no longer perpendicular to the helical axis
Also a right-handed helix
A-DNA is formed from B-DNA

Question 13

B-DNA

Answer 13

Most abundant DNA type - the majority of DNA in a cell is B-DNA

Question 14

Z-DNA

Answer 14

Left-handed helix
Formation is generally unfavourable, but is promoted by certain conditions e.g. negative supercoiling, high salt, some cations
Transient structure occasionally induced by biological activity and then quickly disappears - has made it difficult to characterise/study

Question 15

DNA polymerase

Answer 15

Extends primer in a 5’ to 3’ direction

Cannot function without a primer

Question 16

Primer

Answer 16

Short piece of ssDNA

18 nucleotides long (but can be longer)

Question 17

DNA synthesis schematic

Answer 17

Draw

Question 18

How many DNA molecules are produced after n rounds/cycles of PCR?

Answer 18

2^n

Question 19

Applications of PCR

Answer 19

Forensics e.g. amplifying crime scene DNA
Amplification of ancient DNA
Synthetic biology i.e. making new genes and making the genomes of new organisms
Sequencing genomes i.e. determining the order and identity of bases in a DNA strand

Question 20

How many primers are needed for PCR

Answer 20

2

1 forward and 1 backward

Question 21

Base sequence of forward primer

Answer 21

First 18 bases of ‘top strand’

5’ left and 3’ right

Question 22

Base sequence of backward primer

Answer 22

Complement of the last 18 bases of the top strand

3’ left and 5’ right

Question 23

Problems with PCR

Answer 23

Primer dimers - can occur if the forward primer has 5’ AGC and the backward primer has 3’ TCG
Self-complementary primers - can loop round

Question 24

Chemical synthesis of DNA

Answer 24

i.e. for making primers
Phosphoramidite method
Draw

Question 25

Notes on the chemical synthesis of DNA

Answer 25

Solid phase
Automated
Excess reagents washed away
Can only produce up to ~200-mers (linear synthesis so the yield decreases with each step)
Purification by HPLC or polyacrylamide gel
Possible to modify the 5’ or 3’ end e.g. adding a fluorescent tag

Question 26

PCR scheme

Answer 26

Draw

Question 27

What does a PCR reaction mixture contain?

Answer 27

Target DNA (>= 100 molecules)
Primers
dNTPs (nucleotide triphosphate)
DNA polymerase from Thermus aquaticus
Buffer (pH 7-8)

And either
A layer of mineral oil on top of the mixture
Or
Use a machine with a hot bonnet = heated surface

These will both stop the mixture from evaporating

Question 28

Analysis of PCR reaction mixture

Answer 28

1. Add 30 ml water to conical (Erlenmeyer) flask
2. Add agarose to the water (enough for a 1-3 % gel - approx. 0.6 g)
3. Add a plug of cotton wool into the top of the flask
4. Microwave the mixture - becomes a hot clear solution
   [can also add EtBr/dye of choice at this stage after microwaving]
5. Pour into gel apparatus ready for agarose gel electrophoresis

Question 29

Agarose gel electrophoresis

Answer 29

Very important technique for sizing DNA

Sorts pieces of dsDNA according to size

Question 30

Agarose gel electrophoresis procedure

Answer 30

1. A well is formed in the gel from the comb
2. To the well is added: 5 uL PCR mix, sucrose (helps mixture to sink to the bottom of the well because it is dense) and 2 blue dyes
3. DNA is then sorted/separated out by size - smallest fragments migrate the fastest towards the anode and the largest fragments migrate the slowest
4. Once the gel has run (approx. 30 mins), it is visualised using UV light - this is possible due to the presence of ethidium bromide
5. Can compare the bands to the ‘marker’ lane, which contains bands of DNA of known size

Question 31

2 blue dyes used for agarose gel electrophoresis

Answer 31

Xylene cyanol ff (migrates slowly)
Bromophenol blue (migrates faster)

Question 32

Purpose of blue dyes in agarose gel electrophoresis

Answer 32

Allows you to follow the PCR mixture as it passes through the gel

Question 33

Cathode

Answer 33

Negative end

Question 34

Anode

Answer 34

Positive end

Question 35

Properties of ethidium bromide

Answer 35

(Draw)
Planar, so can slot between bases
Positively charged, so is attracted to the negative phosphate groups in DNA
Fluoresces orange after irradiation with UV light
Not really used anymore - use GelRed (orange) or SYBR Safe (green) nowadays (less carcinogenic)

Question 36

Negative control

Answer 36

Essential when carrying out PCR/agarose gel electrophoresis
Basically the PCR but with no DNA
If this then produces a ‘hit’ in the gel, i.e. it produces a fluorescent band, then it highlights there is some contamination