I. DNA & RNA | 17. Function and application of PCR and real-time PCR

Question 1

I. Basics
1. What is Polymerase chain reaction (PCR)?

Answer 1

Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
=> This makes us able to detect a specific gene by using either electrophoresis (for visualization) or it can be done by real- time PCR on the spot.

Question 2

I. Basics
2. What does majority of PRC methods rely on?

Answer 2

The majority of PCR methods rely on the thermal cycling,

Question 3

I. Basics
3. What are the features of thermal cycling?

Answer 3

The majority of PCR methods rely on the thermal cycling, which involves exposing the reaction mixture to cycles of repeated heating and cooling – permitting different temperature-dependent reactions.

Question 4

I. Basics
4. What are the features of Primers?

Answer 4

• Primers containing complementary sequences to the target regions along with a DNA polymerase (after which the method is named) enable selectivity and repeated amplification.
• The primers used in PCRs are artificial, single- stranded DNA (not normal double-stranded RNA as it is in nature).
• The primer binds to its complementary region in the genome => this is how we select which region we want to amplify.

Question 5

I. Basics
5. What is the general mechanism of PCR?

Answer 5

As PCR progresses, the DNA generated is itself used as a template for replication, setting in a motion of chain reaction in which the original DNA template is exponentially amplified.
=> So for each cycle, all the DNA molecules present, perform a semiconservative replication.

Question 6

I. Basics
6. What are the features of Taq polymerase?

Answer 6

Almost all PCR have a heat-stable DNA polymerase – Taq polymerase (an enzyme originally isolated from a thermophilic bacterium).
=> This DNA polymerase enzymatically assembles a new DNA strand from free nucleotides, by using a single-stranded DNA as a template and primers to initiate synthesis of DNA.

Question 7

I. Basics
7. Can DNA polymerase start the synthesis itself?

Answer 7

As mentioned, DNA polymerases cannot start the synthesis of a new strand (can only continue elongation of an existing strand), therefore, we use primer molecules to indicate the template that we want to replicate.

Question 8

I. Basics
6. What are the features of Taq polymerase?

Answer 8

Almost all PCR have a heat-stable DNA polymerase – Taq polymerase (an enzyme originally isolated from a thermophilic bacterium).
=> This DNA polymerase enzymatically assembles a new DNA strand from free nucleotides, by using a single-stranded DNA as a template and primers to initiate synthesis of DNA.

Question 9

II. PCR procedure (thermocycle)
1. What are the 3 phases of thermocycle?

Answer 9

Thermocycle of a polymerase chain reaction (PCR) can be divided into 3 phases:
1. Denaturation (95 degrees)
2. Annealing (55 degrees)
3. Elongation (72 degrees)

Question 10

II. PCR procedure (thermocycle)
2. What happen in phase 1: Denaturation (95 degrees) of a thermocycle?

Answer 10

• Double-stranded DNA is heat denatured at 95 degrees (45s)
• Hydrogen bonds are broken between the complementary bases => get single-stranded templates of DNA

Question 11

II. PCR procedure (thermocycle)
3. What happen in phase 3: Annealing: (55 degrees)?

Answer 11

• Reaction temperature is lowered to 50-72 degrees (30 – 60s), allowing annealing of primers to each of the ssDNA templates
• 2 primers are needed, one for each of the 2 ssDNA complements containing target reaction mixture (forward + reverse)
• The primers are short single-stranded sequences that will attach at the 3’end of each strand – they mark the borders of the region to be amplified (starting point for DNA polymerase)
• It is important that the temperature is low enough to allow hybridization of the primer to the strand – so we can achieve stable H-bonds between the bases

Question 12

II. PCR procedure (thermocycle)
4. What happen in phase 3: Elongation (72 degrees)?

Answer 12

• Elongation of primers occurs at 72 degrees (corresponds to optimal temperature of the heat-stable Taq polymerase)
• Amplification of DNA by DNA polymerase: synthesize a new DNA strand complementary to the DNA template strand by adding free nucleotides from the reaction mixture (in the 5’ -> 3’ direction)
• Amount of amplified product is determined by primers, nucleotides and the lifetime of DNA polymerase

Question 13

II. PCR procedure (thermocycle)
5. Explain the result after 1st and 2nd cycle of the PCR (and future cycles)?

Answer 13

1. After each cycle, the DNA sequence is doubled. Formula for calculating how many copies we have is 2n, where n = number of cycles. Usually this thermocycle is repeated around 40 times = 40 cycles, therefore 240 number of copies
2. During the 2nd cycle of the PCR (and future cycles), when the DNA synthesis is carried out, it will not stop at the primers on the original template strand
   - The number of the long original template strands will increase linearly (in each cycle there will be 2 new original long strands)
   - But the number of new synthesized strands will increase exponentially

Question 14

II. PCR procedure (thermocycle)
6A. What are the 5 components of reaction mixture of PCR?

Answer 14

• DNA-template: 1-100ng, DNA to be investigated/amplified
• Primer (x2): determines which segment is to be amplified
• dNTP: (deoxy ribose nucleotide triphosphate) building blocks of the new strands
  (dATP, dCTP, dGTP, dTTP)
• DNA polymerase: synthesizes the new DNA chains (thermostable – will not be denatured)
• Buffer: provides optimal environment for the DNA-
  dependent DNA polymerase (last to be added)

Question 15

II. PCR procedure (thermocycle)
6B. What is the role of DNA template in reaction mixture?

Answer 15

1-100ng, DNA to be investigated/amplified

Question 16

II. PCR procedure (thermocycle)
6C. What is the role of Primer in reaction mixture?

Answer 16

Primer (x2): determines which segment is to be amplified

Question 17

II. PCR procedure (thermocycle)
6D. What is the role of dNTP in reaction mixture?

Answer 17

dNTP: (deoxy ribose nucleotide triphosphate) building blocks of the new strands
(dATP, dCTP, dGTP, dTTP)

Question 18

II. PCR procedure (thermocycle)
6E. What is the role of DNA polymerase in reaction mixture?

Answer 18

DNA polymerase: synthesizes the new DNA chains (thermostable – will not be denatured)

Question 19

II. PCR procedure (thermocycle)
6F. What is the role of Buffer in reaction mixture?

Answer 19

Buffer: provides optimal environment for the DNA-
dependent DNA polymerase (last to be added)

Question 20

III. What are the applications of PCR?

Answer 20

Question 21

IV. Gel electrophoresis
1. What is Gel electrophoresis?

Answer 21

Electrophoresis is a technique in which charged macromolecules (RNA, DNA, proteins, carbohydrates) are separated according to their physical properties.

Question 22

IV. Gel electrophoresis
2. What is separation of molecules in gel electrophoresis based on?

Answer 22

Separation is based on the fact that the charged components of a solution migrate at different velocity under the action of an electric field. Separation by size:
- Smaller molecules -> smaller resistance -> larger mobility -> longer traveling distance (faster)
- Larger molecule -> larger resistance -> less mobility -> shorter traveling distance (slower)
=> The pore size of the gel determines the travel speed of the molecules

Question 23

IV. Gel electrophoresis
3. What is the role of Intercalator dye?

Answer 23

Intercalator dye allows us to generate fluorescent light (by using UV light), that allows us to visualize the DNA in the gel. Separation by DNA marker
- DNA is negatively charged (due to phosphate group), will go towards the positive pole

Question 24

IV. Gel electrophoresis
4. How does DNA molecule move in gel electrophoresis? Why?

Answer 24

DNA is negatively charged (due to phosphate group), will go towards the positive pole

Question 25

V. Real-time PCR
1. What is the difference between conventional PCR and real-time PCR?

Answer 25

• In conventional PCR, the amplified DNA product (amplicon) is detected in an end- point analysis
• In contrast, applying a real-time PCR, the accumulation of amplification product is measured as the reaction proceeds – in real time = we get the result during the PCR and not after it

Question 26

V. Real-time PCR
2. How does real-time PCR work?

Answer 26

• Real-time detection of PCR products is made possible by the inclusion of a fluorescent reporter molecule in each reaction, that yields increased fluorescence with an increasing amount of product DNA.
• Specialized thermal cyclers equipped with fluorescence detection modules are used to monitor the fluorescence signal as amplification occurs.
• The measured fluorescence is proportional to the total amount of amplified DNA product (amplicon); the change in fluorescence over time is used to calculate the amount of amplicon produced in each cycle.

Question 27

V. Real-time PCR
3. What is the role of real-time PCR?

Answer 27

With real-time PCR we are able to determine the template DNA both relative and absolute:
- Relative: where we compare the DNA with one another – the aim is to identify the differences between them. F.ex: to identify tumor cells in the tissue (qualitative)
- Absolute: the quantitative determination of the DNA template – identify the exact number of DNA in molecules of grams (quantitative)

Question 28

V. Real-time PCR
4. What are the applications of real-time PCR?

Answer 28

1. Gene expression analysis
2. “Gene dosage”, CNV (copy number variation) analysis - large scale repeat variations
3. SNP genotyping
4. Identification of novel polymorphisms (melting curve analysis)