lecture 2 Flashcards

1
Q

where is most DNA found?

A

right helix

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

what is polymerase chain reaction (PCR)?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

what does PCR produce?

A

new strand of DNA complimentary to the original template strand

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

how does replication occur in prokaryotes?

A

-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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

what is needed for replication in eukaryotic cells?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

what does the alpha polymerase do?

A

initiates the process of leading strand

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

what does the epsilon polymerase do?

A

replicates the lagging strand

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

what does the delta polymerase do?

A

takes over from alpha polymerase, produces the end product

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

why do we use PCR?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

why does PCR require no added energy?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

what occurs in the DNA synthesis reaction?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

what is in the reaction?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

what do the nucleotides add to the structure?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

what is the function of taq polymerase?

A

-accurately copies DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

why use taq polymerase?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

what are the region of the enzyme?

A

synthesis, proof reading and primer removal

17
Q

what occurs in the synthesis region?

A

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

18
Q

what occurs in the proof reading region?

A

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

19
Q

what are the characteristics needed for a primer?

A

-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

20
Q

why is magnesium used?

A

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

21
Q

where does the magnesium bind to?

A

-active site and enables the binding of a protein

22
Q

what is the purpose of the buffer?

A

-promotes the annealing of primers to the DNA

23
Q

what are the three stages of the PCR reaction?

A

-denaturation
-annealing
-elongation

24
Q

What happens at 92 C in PCR?

A

DNA hydrogen bonds between double strands break apart

25
What happens at 55 C in PCR?
primers anneal
26
What happens at 72 C in PCR?
extension of bases
27
what direction does taq polymerase run?
5' to 3'
28
how are PCR products detected?
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