PCR

Question 1

What is PCR?

Answer 1

Polymerase Chain Reaction
* Uses an enzyme (polymerase) to read a single chain on DNA and get the relevant nucleotides from the environment to make more
* Scaling up the amount of chains so there is enough material to do analysis

Question 2

What are the steps involved in PCR?

Answer 2

1. Start with a double stranded template of DNA
2. Heat up to 94°C to denature and helicase split the two strands
3. Then hydridise primers at 64°C - add primers to one end of the DNA (3’ to 5’ end)
4. Use polymerase, starting on the primer, to attach nucleotides from the environment to artificially replicate DNA at 72°C
5. Now we have two double stranded DNA repeats that are exact copies of the template
6. Repeat

Question 3

What can we use PCR to look for?

Answer 3

• Identify genes in <1 week
• Detect mRNAs = gene expression
• Hereditary diseases
• Identify viruses or microbes
• Site-detection mutagenesis
• Paleobiology
• Familial relationships
• Forensic investigations

Question 4

Why are primers used for PCR?

Answer 4

• polymerase can only add bases to pre-existing strands
• designed to target the edge of STRs (highly variable part of human DNA)

Question 5

What does primer concentration determine?

Answer 5

the maximum yield of product - used up in each cycle

Question 6

What are dNTPs?

Answer 6

building blocks of new DNA

Question 7

What are the 4 dNTPs?

Answer 7

• dATP
• dCTP
• dGTP
• dTTP

Question 8

How do dNTPs form new bonds?

Answer 8

have an extra OH bond on the deoxyribose sugar
* allows for the bonding to phosphate groups
* builds off the 3’ end of DNA

Question 9

What is in the buffers when doing PCR?

Answer 9

• salts to stabilise of phosphate groups
• needed for hybridisation and extension

Question 10

What happens during the initalisation part of the PCR process?

Answer 10

• ensures template DNA is fully suspended and properly denatured
• important if template is long
• hot to activate polymerase

Question 11

What happens during the denaturation stage of the PCR process?

Answer 11

• splits double stranded DNA into single standed DNA
• higher temp makes DNA more stable in single strands rather than double

Question 12

What happens during the annealing stage of the PCR process?

Answer 12

• primers bind to template trands
• temperature is lower
• primer binds over complementary template because of high conc
• polymerase will also bind but not proceed yet

Question 13

What happens during the extension stage of the PCR process?

Answer 13

• synthesis of complementary strand
• temperature is optimised for activity of enzyme

Question 14

What happens during the cycling of the PCR process?

Answer 14

• each cycle doubles DNA conc
• too few = not enough amplification
• too many = limited by dNTP conc
• too many will lead to truncated products

Question 15

What happens during the final extension and hold stage of the PCR process?

Answer 15

• ensures all strands are finished
• reduces truncated products
• then cool right down for final hold (most stable temp for DNA so wont degrade) and storing

Question 16

How is a primer dimer formed?

Answer 16

• poor choice of primer sequence
• too much primer added

Question 17

What is a primer dimer?

Answer 17

primer binds together rather than onto the strands

Question 18

What does it mean if there is no amplification?

Answer 18

• primer too concentrated
• dNTPs degraded by freezing
• template has degraded
• annealing temp too high
• you forgot somethign

Question 19

What does it mean if there is non-specific amplification?

Answer 19

• contamination
• annealing temp too high

Question 20

What does it mean if there is a weak amplification?

Answer 20

• conc too low
• not enough cycles
• annealing time too short

Question 21

What is reverse transcript PCR?

Answer 21

• RNA
• bind primer
• reverse transcription
• denature to form cDNA
• add primer to cDNA
• PCR to make copies of cDNA

Question 22

What is quantitative PCR?

Answer 22

• detect fluorescene in real time
• conc of DNA product proportional to fluorescence
• polymerases will digest the probe stand while forming complementary strand
• probe stand contains fluorophore and quencher
• while the probe is still on the template there will be no fluorescence because the quencher is too close to the fluorophore
• when polymerase digests the probe it will release the fluorophore and quencher so fluorescence will happen

Question 23

What is digital PCR?

Answer 23

• same as qPCR but on a larger scale
• more sensitive measurement of amount