Intro and PCR Flashcards
Direction of DNA
5’ to 3’
Charge on phosphate group at physiological pH
Negative
What does a nucleotide consist of?
Base
Sugar
Phosphate group
How long is a typical DNA strand?
10 - 10^9 nucleotides long
Structure of bases
Draw
Handedness of DNA double helix
Right handed
Width of DNA double helix (i.e. helix diameter)
2 nm (20 A)
Length of 1 helical turn
3.4 nm (10 base pairs)
DNA strands are…
…antiparallel
Base pairs are…
…perpendicular to the helical axis
Hydrogen bonds between bases
Draw
A-DNA
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
B-DNA
Most abundant DNA type - the majority of DNA in a cell is B-DNA
Z-DNA
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
DNA polymerase
Extends primer in a 5’ to 3’ direction
Cannot function without a primer
Primer
Short piece of ssDNA
18 nucleotides long (but can be longer)
DNA synthesis schematic
Draw
How many DNA molecules are produced after n rounds/cycles of PCR?
2^n
Applications of PCR
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
How many primers are needed for PCR
2
1 forward and 1 backward
Base sequence of forward primer
First 18 bases of ‘top strand’
5’ left and 3’ right
Base sequence of backward primer
Complement of the last 18 bases of the top strand
3’ left and 5’ right
Problems with PCR
Primer dimers - can occur if the forward primer has 5’ AGC and the backward primer has 3’ TCG
Self-complementary primers - can loop round
Chemical synthesis of DNA
i.e. for making primers
Phosphoramidite method
Draw
Notes on the chemical synthesis of DNA
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
PCR scheme
Draw
What does a PCR reaction mixture contain?
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
Analysis of PCR reaction mixture
- Add 30 ml water to conical (Erlenmeyer) flask
- Add agarose to the water (enough for a 1-3 % gel - approx. 0.6 g)
- Add a plug of cotton wool into the top of the flask
- Microwave the mixture - becomes a hot clear solution
[can also add EtBr/dye of choice at this stage after microwaving] - Pour into gel apparatus ready for agarose gel electrophoresis
Agarose gel electrophoresis
Very important technique for sizing DNA
Sorts pieces of dsDNA according to size
Agarose gel electrophoresis procedure
- A well is formed in the gel from the comb
- 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
- DNA is then sorted/separated out by size - smallest fragments migrate the fastest towards the anode and the largest fragments migrate the slowest
- Once the gel has run (approx. 30 mins), it is visualised using UV light - this is possible due to the presence of ethidium bromide
- Can compare the bands to the ‘marker’ lane, which contains bands of DNA of known size
2 blue dyes used for agarose gel electrophoresis
Xylene cyanol ff (migrates slowly) Bromophenol blue (migrates faster)
Purpose of blue dyes in agarose gel electrophoresis
Allows you to follow the PCR mixture as it passes through the gel
Cathode
Negative end
Anode
Positive end
Properties of ethidium bromide
(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)
Negative control
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
