Study sheet: PCR and Plasmid Cloning

📋 Course Outline

1. PCR Principle and Components
2. PCR Cycle and Amplification
3. Melting Temperature and Primers
4. PCR Reaction Optimization
5. Plasmid Vectors and Selection
6. Restriction Digestion and Cloning

📖 1. PCR Principle and Components

🔑 Key Concepts & Definitions

• Polymerase Chain Reaction : a fast and inexpensive technique that exponentially amplifies a selected target DNA molecule through repeated heating and cooling cycles

★ Must-know

• PCR applications include:

  • genotyping
  • cloning
  • mutation detection
  • medical diagnostics
  • sequencing

• A basic PCR requires:

  • a DNA template containing the target region
  • heat-resistant Taq DNA polymerase
  • two primers with free 3'OH groups
  • dNTPs
  • buffer
  • bivalent cations usually MgCl2
  • a PCR machine

Further detail

• PCR is credited to Kary Mullis in 1983, and he received the Nobel Prize in Chemistry with Michael Smith for this invention in 1993.

📖 2. PCR Cycle and Amplification

★ Must-know

• Each PCR cycle consists of denaturation at 95°C, annealing at 50–65°C, and extension at 72°C.

• Under efficient reaction conditions, PCR can amplify one or a few starting copies of the target region to billions of copies.

Further detail

• PCR cycles are usually repeated 20 to 40 times depending on the number of starting copies.

• The first, second, and third PCR cycles produce two, four, and eight double-stranded DNA molecules, respectively.

💡 Memory Hook

Denaturation → annealing → extension

📖 3. Melting Temperature and Primers

🔑 Key Concepts & Definitions

• Melting Temperature : the temperature at which 50% of duplex DNA dissociates into single strands

★ Must-know

• The Tm indicates duplex stability, and PCR specificity depends strongly on the Tm of the primers.

📐 Formula — The commonly used primer formula is $Tm = 4(G+C) + 2(A+T)$, and 5 degrees must be subtracted when setting up the PCR machine: $Tm = 4(G+C) + 2(A+T) - 5$.

• PCR primers are synthetic DNA oligonucleotides approximately 15 to 30 bases long that bind complementary sequences flanking the target region.

Further detail

📌 Primer melting temperatures should be between 55 and 70°C, with no more than 5°C difference between the two primers.

📖 4. PCR Reaction Optimization

★ Must-know

• Template DNA may be:
  • genomic DNA
  • complementary DNA
  • plasmid DNA
  • a PCR product

📌 Higher template amounts increase the risk of nonspecific amplification, whereas lower amounts reduce PCR yields.

📌 In a typical 50 µL reaction, 1 to 2 units of DNA polymerase are sufficient, but increasing the enzyme amount may improve amplification in the presence of inhibitors while producing nonspecific products at higher concentrations.

Further detail

• In a 50 µL PCR, 0.1–1 ng of plasmid DNA or 5–50 ng of genomic DNA may be sufficient as starting template amounts.

• Taq DNA polymerase has a half-life of about 40 minutes at 95°C, incorporates nucleotides at about 60 bases per second at 70°C, and can amplify products of about 5 kb.

📌 PCR primers should have unique binding sites near the target and should not have large complementarities with each other or self-complementarity that causes secondary structures and self-priming.

• PCR buffer usually has a pH between 8.0 and 9.5, often stabilized by Tris-HCl, and KCl promotes primer annealing for Taq polymerase.

• Common PCR additives and their final concentrations are:

  • DMSO at 1–10%
  • glycerol at 5–20%
  • formamide at 1.25–10%

💡 Memory Hook

More template or polymerase can increase yield but also nonspecific products

📖 5. Plasmid Vectors and Selection

🔑 Key Concepts & Definitions

• Plasmid Vector : designed to clone DNA fragments generally smaller than 10,000 base pairs in bacteria

★ Must-know

• Plasmid vectors contain at least one bacterial origin of replication and one antibiotic resistance gene, such as ampicillin, kanamycin, or tetracycline resistance.

📌 The plasmid origin of replication allows plasmids to replicate independently of bacterial chromosome replication.

📌 Applying the corresponding antibiotic to bacterial cultures kills bacteria that do not contain the plasmid.

Further detail

• The Bluescript pBSKSII+ plasmid is 2961 bp long and contains an ampicillin-resistance gene, a ColE1 origin, and multiple cloning sites with unique restriction sites.

💡 Memory Hook

Antibiotic selection → survival of plasmid-containing bacteria

📖 6. Restriction Digestion and Cloning

🔑 Key Concepts & Definitions

• Restriction Enzyme : recognizes a specific short DNA sequence and cleaves DNA at that site to produce compatible ends

★ Must-know

• Restriction digestion can generate:

  • 5' cohesive ends
  • blunt ends
  • 3' cohesive ends

• Molecular cloning produces recombinant DNA molecules that are used to transform a host organism in which they are replicated.

• The DNA fragment and linearized plasmid must have compatible ends before the fragment is inserted into the vector through phosphodiester bonds to form a recombinant plasmid.

Further detail

• Restriction enzymes may recognize sites of:

  • 4 base pairs
  • 6 base pairs
  • 8 base pairs
  • 10 base pairs
  • 12 base pairs

• EcoRI recognizes a 6-base-pair palindromic sequence and generates 5' cohesive ends.

• NotI recognizes an 8-base-pair palindromic sequence, making its recognition site rarer than EcoRI's 6-base-pair site.

💡 Memory Hook

Digestion → ligation → transformation → analysis

📊 Synthesis Tables

PCR Cycle Steps

Step	Temperature	Function
Denaturation	95°C	Separates double-stranded DNA
Annealing	50–65°C	Allows primers to bind
Extension	72°C	Taq polymerase synthesizes DNA