Unit 2: The polymerase chain reaction

Question 1

What is PCR ?

Answer 1

A method used to amplify large amounts of DNA from a small amount of starting material

Question 2

What type of DNA polymerase is used in PCR ?

Answer 2

Taq DNA polymerase

Question 3

What is Taq made from ?

Answer 3

Bacteria that lives at high temps in hot springs and deep sea vents

Question 4

What is an amplicon ?

Answer 4

A piece of DNA that is the product of amplification

Question 5

What is a forward strand ?

Answer 5

5’ to 3’

Question 6

What is a reverse strand ?

Answer 6

3’ to 5’

Question 7

Explain the process of PCR (4)

Answer 7

1) Heat to 94 degrees to denature DNA strands
2) Cool to 50-60 degrees to allow primers to anneal
3) Heat to 72 to allow for elongation of new strands (Addition of nucleotides)
4) 2 New strands of DNA generated (At end of first cycle)

Question 8

What is an upstream primer ?

Answer 8

Short sequence of nucleotides used to initiate the amplification of a specific DNA segment

Question 9

What is the reverse primer in respect to the reverse strand ?

Answer 9

The reverse primer is complementary to the reverse strand but it needs to be written as 5’ to 3’ so it is reversed

Question 10

Can primers also anneal to products of the PCR ?

Answer 10

Yes they can

Question 11

How many cycles of PCR are there typically ?

Answer 11

30-40 cycles

Question 12

What is included in a PCR reaction mix (7) ?

Answer 12

1) dNTPs
2) Reaction buffer containing Mg2+ ions
3) Forward primer
4) Reverse primer
5) Taq polymerase
6) Template DNA
7) dH2O

Question 13

What are 5 things to consider when designing a primer for PCR ?

Answer 13

1) 18-22 nucleotides in length
2) GC content of 40-60% (More GC to AT)
3) Tm (melting temp) of 55-65C
4) Both primers should have a similar Tm
5) Primers should ONLY have similarity to site being amplified

Question 14

How long is the initial denaturation stage ?

Answer 14

2-5 mins

Question 15

How long is the denaturation stage ?

Answer 15

30-60 seconds

Question 16

How long is the annealing stage ?

Answer 16

30-60 seconds

Question 17

How long is the elongation stage ?

Answer 17

30-60 seconds

Question 18

How long is the final elongation stage ?

Answer 18

5 mins

Question 19

What 3 sections of the PCR programme needs to be repeated 25-35 times ?

Answer 19

• Denaturation
• Annealing
• Elongation

Question 20

How can the total number of DNA produced be calculated ?

Answer 20

2^ number of cycles

Question 21

How can the amplification of DNA in PCR be described ?

Answer 21

As being exponential

Question 22

What are the 2 stages of PCR ?

Answer 22

• Exponential phase
• Plateau phase

Question 23

What is the exponential phase ?

Answer 23

100% PCR efficiency- amount of DNA doubles each cycle

Question 24

What is the plateau phase ?

Answer 24

PCR is less than 100% efficient

Question 25

Why does the actual yield from a PCR plateau not carry on as theoretical suggests ?

Answer 25

- Damages Taq and used up available dNTPs and primers - Enzyme degrades - Run out of reagents and nucleotides

Question 26

How can PCR products be analysed ?

Answer 26

By gel electrophoresis

Question 27

What is one problem with Taq polymerase ?

Answer 27

It lacks 3'-5' exonuclease proofreading activity so it can't reverse and correct errors if it incorporates the wrong base

Question 28

What are 4 uses of PCR ?

Answer 28

1) Amplification for cloning 2) Amplification for DNA sequencing 3) Detection of mutations 4) Detection of relatedness

Question 29

What is RT-PCR ?

Answer 29

Reverse transcription PCR

Question 30

What is RT-PCR ?

Answer 30

- Allows for detection of gene expression in cells and tissues - Copy of mRNA is made into cDNA - cDNA is used as template strand

Question 31

What 3 primers are used in RT-PCR ?

Answer 31

1) Gene specific 2) Oligo dT 3) Random hexamers

Question 32

Why is RT-PCR used in virus testing ?

Answer 32

- They are highly sensitive so can detect copies 17 days after infection

Question 33

What is qPCR ?

Answer 33

Real time or quantitative PCR

Question 34

What is the function of qPCR ?

Answer 34

It allows us to accurately determine the number of copies of a DNA sequence in starting material

Question 35

What is SYBR ?

Answer 35

A green dye that only binds to double stranded DNA

Question 36

What is the SYBR green method ?

Answer 36

- As the amount of PCR product increases each cycle so does the amount of SYBR green dye bound - Laser is fired into sample and floresence is recorded

Question 37

What are 4 uses of qPCR ?

Answer 37

1) Quantative measurement of gene expression between samples 2) Analysis of copy number variations 3) Quantification of virus numbers in an individual or sample 4) Microbiological monitering of food/water