Molecular Biology Techniques

Question 1

Polymerase Chain Reaction (PCR) Test

Purpose

Answer 1

Amplify a target DNA sequence

Question 2

Polymerase Chain Reaction (PCR) Test

Key Traits

Answer 2

Repetition of 20-30 times
a. Amplification of target sequence to 2^{n</sub> copies
b. Highly specific amplification of target DNA
c. Amplification is completed in a very short duration}

Question 3

Polymerase Chain Reaction (PCR) Test

Key Principal

Answer 3

Complementary Base Pairing between the target DNA sequence and PCR Primer

Question 4

Polymerase Chain Reaction (PCR) Test

Components

Answer 4

1. DNA Sample
2. PCR Primers
3. Free Deoxyribonucleotides (dNTPs)
   a. dATP
   b. dTTP
   c. dCTP
   d. dGTP
4. Heat-resistant Taq Polymerase
5. PCR Buffer

Question 5

Taq Polymerase

Key Characteristic

Answer 5

Thermo-stable DNA polymerase with the ability to withstand high temperatures without Denaturation

Question 6

Taq Polymerase

Purpose

Answer 6

Catalyse the formation of the Phosphodiester (PDE) Bond to extend the DNA primers during amplification of the target sequence

Question 7

PCR Buffer

Purpose

Answer 7

1. Contains pH buffer that maintains optimal pH
2. Contains salts that provide the environment for the reaction
3. Contains MgCl₂ that undergoes dissolution into Mg²⁺ which is needed as a co-factor of Taq polymerase

Question 8

PCR Primers

Components

IE. Forward/ Reverse Primers

Answer 8

2 sets of single-standed oligonucleotide primers, roughly 20 DNA bases long that are complementary to the 3’- end of the target DNA sequence on the template strand.

Present in large excess

Question 9

PCR Primers

Purpose

Answer 9

1. Anneal to target DNA sequence through H-bond formation upon CBP
2. Provides free 3’-OH group for Taq Polymerase to add free dNTPs
3. Specifies the target DNA sequence for amplification

Question 10

PCR Buffer

Components

Answer 10

1. Detergent
2. pH Buffer/ Buffering Salts
3. MgCl₂

Question 11

Polymerase Chain Reaction (PCR) Test

Procedure

Answer 11

1. Denaturation: Reaction mixture heated to 95°C for 30s in a Thermocycler
2. Primer Annealing: Reaction mixture cooled to 55°C for 60s
3. Primer Extension: Reaction mixture heated to 72°C for ~120s

Question 12

Polymerase Chain Reaction (PCR) Test

Significance of Procedure

Answer 12

1. Denaturation
   a. H-bond between double-stranded DNA (dsDNA) broken
   b. Single-stranded DNA (ssDNA) formed upon denaturation
2. Primer Annealing
   a. Forward & reverse Primers anneal to 3’- end of ssDNA template through H-bond formation upon CBP
3. Primer Extension
   a. Taq Polymerase extends both primers in the 5’ to 3’ direction
   b. Target sequence amplified

Question 13

Polymerase Chain Reaction (PCR) Test

Advantages

Answer 13

1. Highly sensitive & specific
   a. Specific target sequence amplified with minute amounts at the start
2. Very fast & efficient
   a. Capable of being automated
   b. 20-30 cycles takes 2-3 hours
3. Taq Polymerase is highly thermo-stable
   a. Synthesis of DNA strands occurs in high temperatures of 72°C without denaturation
   b. Can without and be reused for multiples without denaturation

Question 14

Polymerase Chain Reaction (PCR) Test

Limitations

Answer 14

1. Lack of proof-reading capabilities
   a. Absence of 3’ to 5’ exonuclease activity
   b. Inability to correct incorrect addition of dNTP
   c. 1/10000 chance of error
   (d. Mutations)
2. Limited amounts and size of PCR products
   a. Ideal amount of 0.1 to 5.0kb of DNA products
   b. Longer PCRs have a higher chance of errors
3. Pre-requisite of target DNA sequence

1 kb = 1000 bases

Question 15

DNA Replication vs. PCR Test

Similarities

Answer 15

1. DNA as template
2. Requirement of dNTPs
3. Formation of PDE bond

Question 16

DNA Replication vs. PCR Test

Differences

Answer 16

1. Enzyme that catalyses PDE
2. Primer type
3. Number of primers
4. Section of DNA to be replicated
5. Presence of leading/ lagging strand
6. Temperature
7. Method of ‘unzipping’