Molecular Biology Lecture Review (recording)

Overview of PCR Properties

• Discussion focuses on several key properties of primers and their roles in PCR (Polymerase Chain Reaction).

• Topics include primer length, melting temperature (TM), carbon diameters, and carbon models.

Primer Length

• Importance of Primer Length:

  • If a primer is too short, it may bind to multiple sites on long template DNA due to random chance, which can lead to non-specific amplification.

  • The probability of finding a complementary sequence in template DNA is calculated as 4^n, where n is the number of bases in the primer.

  • Example: A primer of length 5 has a binding probability of 4^5 = ~1 in 1024 bases; thus, it might find a match every ~1,024 base pairs.

  • A 6 base pair primer has a probability of finding a match every ~4,096 bases (1 in 4,096).

• Optimal Primer Length:

  • Ideal primer length is typically between 18 to 30 bases.

  • A longer primer (e.g., 18 bases) has a significantly lower chance of non-specific binding, as it corresponds to 4^18, an astronomically larger number, thus reducing likelihood of match within template DNA.

Melting Temperature (TM)

• Definition of TM:

  • The temperature at which 50% of DNA strands are single-stranded and 50% are double-stranded.

  • TM can be affected by primer length and composition, which can impact the annealing temperature in PCR.

• Calculating TM:

  • General formula: TM = 2(A+T) + 4(G+C), where A, T, G, and C are the counts of respective nucleotides.

• Annealing Temperature:

  • Determined as 2–4 °C below the TM of the primer with the lowest TM.

  • Importance of maintaining an annealing temperature that allows for specific binding without allowing mismatches.

Issues with Too Long Primers

• Long primers can be highly specific, but may also:

  • Fail to bind adequately if too specific.

  • Increase the difficulty in maintaining appropriate TM during PCR.

Primer Dimer Formation

• Definition:

  • Occurs when two primers bind to each other rather than to the target DNA.

  • Can result in non-specific amplification and reduce efficiency.

• Implication:

  • Can create confusion in PCR results; presence of primer dimers can result in visible bands in gel electrophoresis, complicating analysis.

Intramolecular Base Pairing

• Occurs when a single primer has complementary sequences within itself, causing it to fold and reducing accessibility for binding to template DNA.

  • This can hinder the efficiency of PCR amplification since the active three-prime end must be available for DNA polymerase to act.

Designing Effective Primers

• Consensus Sequences:

  • When the target sequence is unknown, researchers align known sequences from similar organisms to identify a consensus sequence for primer design.

• Degenerate Primers:

  • Designed based on the amino acid sequence of the protein, accounting for the degeneracy of the genetic code, enabling binding at various codons for a given amino acid.

PCR Product Cloning Methods

• TA Cloning:

  • Taq polymerase adds an 'A' overhang to PCR products, allowing for easy insertion into plasmids with complementary 'T' overhangs.

• Use of Ligase:

  • DNA ligase is typically used to seal the gaps between insert and plasmid DNA, creating stable recombinant plasmids.

• Topoisomerase-Directed Cloning:

  • Topoisomerase can facilitate the insertion of a PCR product into a plasmid efficiently without needing an additional ligation step, enhancing efficiency of the cloning process.

Challenges of PCR Products

• Error Prone Nature:

  • Taq polymerase lacks proofreading capability (3′ to 5′ exonuclease activity); hence, error rates can be significant (up to 1 in 5,000 base pairs).

  • Errors may lead to mutations or premature stop codons in the amplified gene, affecting downstream applications like protein expression.

• Correction Factors:

  • Ensuring the purity of PCR products and validating sequences through subsequent DNA sequencing help identify and eliminate erroneous sequences.