lecture 2

Question 1

where is most DNA found?

Answer 1

right helix

Question 2

what is polymerase chain reaction (PCR)?

Answer 2

-amplification (exact copying) of DNA
-adds bases onto 3’ end

Question 3

what does PCR produce?

Answer 3

new strand of DNA complimentary to the original template strand

Question 4

how does replication occur in prokaryotes?

Answer 4

-enzyme helicase (by using energy) separates the ds DNA to form replication fork
-breaks the Watson crick bonds
-primases attach RNA to open strands to form natural primer
-DNA polymerase attaches to the double strand (at one end)
-slides along 5’ to 3’ of leading strand to produce complimentary strand
-produces one long strand of DNA
-product also produced for complimentary to lagging strand
-primase comes along to add another primer and form small DNA fragments
-ligase comes and ligates strands together forming phosphate backbone
-continuous piece of DNA formed

Question 5

what is needed for replication in eukaryotic cells?

Answer 5

-template DNA
-40 or more proteins
-helicase
-primase (initiation)
-polymerases (Alpha, delta and epsilon)
-nuclease
-ligase
-ssDNA binding proteins
-sliding clamps

Question 6

what does the alpha polymerase do?

Answer 6

initiates the process of leading strand

Question 7

what does the epsilon polymerase do?

Answer 7

replicates the lagging strand

Question 8

what does the delta polymerase do?

Answer 8

takes over from alpha polymerase, produces the end product

Question 9

why do we use PCR?

Answer 9

sensitive, specific, cheap, rapid, robust (DNA is very stable)

Question 10

why does PCR require no added energy?

Answer 10

dNTP is an energy storage molecule so provides the energy for the reaction

Question 11

what occurs in the DNA synthesis reaction?

Answer 11

-hydrolysis of phosphorus dimer at end
-phosphate released and binds to hydroxyl group on previous ribose sugar to form phosphodiester bond

Question 12

what is in the reaction?

Answer 12

template, 2 primers (to prime synthesis), polymerase (copies template of DNA), dNTPs, magnesium (cofactor for DNA polymerase), buffer (maintains pH)

Question 13

what do the nucleotides add to the structure?

Answer 13

strength (phosphate) and specificity (from base) and helps with orientation

Question 14

what is the function of taq polymerase?

Answer 14

-accurately copies DNA

Question 15

why use taq polymerase?

Answer 15

has a high conc at high temperatures- doesn’t denature within the process and temp changes

Question 16

what are the region of the enzyme?

Answer 16

synthesis, proof reading and primer removal

Question 17

what occurs in the synthesis region?

Answer 17

-where protein binds two DNA and synthesises by forming phosphodiester bonds

Question 18

what occurs in the proof reading region?

Answer 18

-makes sure the right nucleotides come and bind at the correct time
-checks if Watson crick bonds are formed accurately

Question 19

what are the characteristics needed for a primer?

Answer 19

-single stranded DNA (oligonucleotide)
-18-24 bases in length (still specific but hybridises quickly)
-high GC content to provide strong attachment and bonds (start and end with GC)
-annealing point/ melting temp of 50-60 degrees
-primer pairs have difference of 5 degrees so can happen in same cycle (efficient)
-primer pairs cant have complimentary region as they’ll bind together

Question 20

why is magnesium used?

Answer 20

-cofactor to maintain activity of primer
-enables activity of the catalysis
-enhances enzymatic activity and supports dna application

Question 21

where does the magnesium bind to?

Answer 21

-active site and enables the binding of a protein

Question 22

what is the purpose of the buffer?

Answer 22

-promotes the annealing of primers to the DNA

Question 23

what are the three stages of the PCR reaction?

Answer 23

-denaturation
-annealing
-elongation

Question 24

What happens at 92 C in PCR?

Answer 24

DNA hydrogen bonds between double strands break apart

Question 25

What happens at 55 C in PCR?

Answer 25

primers anneal

Question 26

What happens at 72 C in PCR?

Answer 26

extension of bases

Question 27

what direction does taq polymerase run?

Answer 27

5’ to 3’

Question 28

how are PCR products detected?

Answer 28

1. run products on agarose gel
   use Intercalating dye to stain DNA to determine size and yield
2. adding ethidium bromide which intercalates between base pairs and fluoresces in UV light