Nucleic Acid Extraction and Amplification Quiz

Nucleic Acid Extraction and Amplification

• Nucleic acid is isolated and purified from the sample, and then the target or nucleic acid of interest is amplified.
• Specific Target Sequence Amplification PCR copies only a very specific sequence of genetic code from a template DNA, targeted by PCR primers.

PCR Process

• PCR consists of three stages: denaturation, annealing, and extension.
• The principle of PCR requires reliable heating and cooling steps, which are achieved using a thermocycler or PCR machine.

Denaturation Stage

• The double-stranded template DNA is denatured by heating to separate the DNA strands.
• Heating to 95°C breaks the hydrogen bonds between complementary bases and hydrophobic stacking interactions, separating the double helix structure of DNA.
• This step generally lasts 15-30 seconds.

Annealing Stage

• The reaction is rapidly cooled to an annealing temperature (50-65°C) to allow the oligonucleotide primers to hybridize to single-stranded template DNA.
• Primers will anneal only to target DNA sequences that are complementary to them, following the rules of base-pair complementarity.
• The temperature is based on the melting temperature (tm) of the primer.

Extension Stage

• Once primers bind to the templates, a DNA polymerase reads the template sequences and adds complementary nucleotides to the 3' ends of the primers.
• The optimum temperature of Taq DNA polymerase is in the range of 70-80°C.
• The extension time depends on the DNA polymerase used and the length of the target DNA sequence to amplify.
• Under optimum conditions, Taq polymerase extends 1000 bases in 1 minute.

PCR Essentials

• 5 critical components are needed to perform PCR:
  • DNA template containing the target sequence to amplify
  • Thermostable DNA polymerase (e.g., Taq enzyme)
  • PCR inhibitors should be eliminated or minimized
• PCR inhibitors can originate from the sample or be introduced during sample processing or nucleic acid extraction.

Primer Requirements

• 2 primers are needed to amplify the target sequence: forward primer and reverse primer.
• Each primer is complementary to a stretch of DNA to the 3' side of the target DNA.
• Primer design and primer specificity are crucial for PCR.

PCR Troubleshooting

• Identifying the location of the target sequence in the DNA template is essential.
• PCR optimization and post-PCR analysis using agarose gel electrophoresis (AGE) are important steps.
• Visualizing PCR results using AGE helps to identify PCR products.

PCR Product

• After 20 cycles, PCR can produce about a million (220) copies of the target.
• The specific sequence will be accumulated in billions of copies (amplicons) at the end of the PCR reaction.