3 Nucleic Acid Amplification

• 3 types of amplification techniques:

  • Target Amplification – specific target DNA is being amplified (e.g., PCR)
  • Probe Amplification – increase the amount of probe molecules, which is the specific target DNA, so they can easily be detected
  • Signal Amplification – it produces more signal; more visual color for us to easily detect
• Polymerase Chain Reaction AKA Molecular Photocopying, is under target amplification. It amplifies the copy of target DNA (specifically small segment of DNA)
• PCR involves in vitro enzymatic replication of DNA

  • Standard PCR → DNA sample only is being replicate (in vivo DNA Replication)
• DNA polymerase which extends and adds up the free nucleotides to produce complementary DNA, is an enzyme used by PCR
• Chain Reaction in PCR is a cycle, usually 30 cycles in 4 hours
• Denaturation is the separation of the two strands of the DNA from one another so that we can create an exact replica of the other strand.
• Normally in our body, denaturation happens because of the actions of our DNA helicase, but in PCR, this happens because of heat, it facilitates denaturation
• Once the nucleic acid have been denatured; this is now the time where primers will be added, it gives specificity to PCR testing because it contains sequences that are similar to DNA template
• After the primers have been added, the next step is the addition of the deoxynucleotide triphosphate, it would be extended due to DNA polymerase (in the form of Taq Polymerase), DNA will be extended properly
• There is an Exponential growth/increase in the number of nucleic acid copies in every cycle of the PCR.
• Kary Mullis is an American biochemist who invented PCR in 1987
• Basic 3 steps of PCR cycle:

  • Denaturation – denature the dsDNA into ssDNA
  • Annealing – addition or binding of primers (primers should bind/hybridize in the template)
  • Extension (aka Elongation/DNA Synthesis) – DNA polymerase extend primers to produce complementary strand; addition of nucleotides to the growing DNA chain by Taq polymerase
• Exponential growth in every cycle of PCR doubles the number of the product molecules.
• Formula for Exponential replication of DNA in PCR

  
• DNA template on the sample DNA that contains the target sequence, which should be known in order to detect them
• By target sequence, we can produce vaccines
• DNA template is retrieved from the the extracted nucleic acid of the patient's sample
• PCR primers uses 1 pair in standard PCR: Forward and Reverse primers

  • Forward: 5’ to 3’ direction
  • Reverse: 3’ to 5’ direction
• Primers have short sequence of nucleotides (20-30 bp) that provides a starting point for DNA synthesis (complementary to the target DNA)

  • Annealing temperature is 2-5°C below the melting temperature of the primers.
• Sequences are based on the database of NCBI

  
• DNA polymerase synthesizes the new strands of DNA complementary to the target sequence and begins synthesizing new DNA strand from the end of the primer
• DNA polymerase have 2 capabilities that make them suitable for PCR:

  • Can generate new strands of DNA using a DNA template and primers
  • in vivo – RNA primers
  • in vitro – DNA primers
  • Heat resistant to maintain the enzyme (DNA polymerase) because it undergoes denaturation process.
• Taq Polymerase is derived from Thermis aquaticus

  • first and most commonly used enzyme
  • other activities: polymerase activity, reverse transcriptase, exonuclease, etc.
• Pfu Polymerase is derived from Pyrococcus furiosu

  • used because of its higher fidelity (has a exonuclease activity)
  • can withstand higher temp than Taq Polymerase
• Pwo Polymerase is derived from Pyrococcus woesei; used for short sequences only
• Tth Polymerase is derived from Thermus thermophilus

  • activities include: reverse transcriptase activity
• Deoxynucleotide triphosphates are nucleotides that have single units of the bases A, T, G, and C; it is an essential "building blocks" for new DNA strands
• Buffer provides a suitable chemical environment for optimum activity and stability of the DNA polymerase
• Buffer is usually usually between 8.0 and 9.5 pH

  It is often stabilized by:

  • Tris-HCl [Tris Hydrochloric acid]
• Thermocyclers are PCR machine

  
• Thermal Denaturation is the first step in a PCR cycle where it needs to denature the dsDNA into two ssDNA for replication done by heating the sample at 90-96°C for 20-60 seconds
• Annealing or Binding is the second step in PCR cycle where temperature is lowered (cool down) between 50-70°C in order for the primers to bind with the template DNA

  • 2 primers will attach to their binding positions on the ssDNA template
  • In general, the optimum annealing temperature is about 3-5°C below the Tm (typically 50-60°C) → this step generally lasts 15 - 60 second
• In annealing, if the temperature is:

  • Too high, it prevents the stability of the hybridization
  • Too low, it leads to non-specific hybridization
• Extension is the third step in PCR cycle where DNA synthesis occurs.

  • Taq Polymerase binds to the template DNA and starts adding nucleotides that are complementary to the first strand.
  • Temperature should be 68-75°C
  • Usually 1 minute extension per cycle
• Exponential Growth in PCR cycle:

  • Cycle is repeated 25-35 times (ave. 30), generally takes 2-4 hours
  • New DNA that is made in one round can serve as template in the next round of DNA synthesis
• A) 90-96

  B) 50-70

  C) 68-75

  D) 20-60

  E) 20-90

  F) 10-60

  
• Reverse-Transcriptase Polymerase Chain Reaction amplifies RNA targets; RNA molecules via reverse transcriptase enzyme is converted to cDNA molecule, then utilized as template sequence for following PCR reaction
• Reverse transcriptase enzyme is the enzyme involved in RT-PCR; it is also a type of polymerase but RNA dependent
• Application of RT-PCR is to detect RNA expression and RNA viruses
• Introns are present in the DNA, but is not present in cDNA