DNA Manipulative Enzymes & DNA Cloning

🧪 What is DNA Cloning?

• Process of manipulating DNA to join genetic material from different organisms

• Creates recombinant DNA

• Enables isolation & amplification of genetic material for study

🧰 What’s Required?

1. Manipulative enzymes

2. Vectors (plasmids, viruses, etc.)

3. Host organisms (usually bacteria)

💰 Why is This Important?

• 60% of enzymes used in industry (e.g. detergents, food) are recombinant

• Therapeutic antibodies market projected at $300B by 2025

• RNA vaccines reached ~$40B in 2021

• CRISPR/Cas9 recently approved — list price: £1.6M per treatment

🧬 Recombinant DNA = Modifiable

We can:

• Change DNA length

• Alter bases

• Remove or add nucleotides/sequences

✂ DNA-Manipulating Enzymes

🧼 Exonucleases

• Remove nucleotides from ends of DNA/RNA

• Act on ssDNA, dsDNA, RNA

• Important for:

  • RNA degradation

  • Proofreading in DNA polymerases

  • Gibson Assembly in cloning

🪓 Endonucleases

• Cut within DNA strands

• Can be specific (restriction enzymes) or non-specific

• Example: DNase I – breaks down extracellular DNA

🔐 Restriction Enzymes (Restriction Endonucleases)

• Natural defense against phages in bacteria

• Sequence-specific cutters (“molecular scissors”)

• Type II enzymes used most in research

• Isoschizomers: different enzymes → same recognition site & cut

🔍 Uses:

• Mutation diagnostics

• DNA fingerprinting (band pattern analysis)

• Plasmid construction

🧷 DNA Ligases

• Join nicks in DNA (especially lagging strand → Okazaki fragments)

• Repair dsDNA breaks

• Consume 2 ATP per linkage

• T4 DNA ligase:

  • Works with sticky/blunt ends, RNA/DNA hybrids

  • High efficiency – ideal for lab use

🧬 Ligation During Restriction Cloning

• Sticky ends produced by restriction enzymes anneal (base pair)

• Ligase seals the phosphodiester backbone

🧠 Factors Affecting Ligation:

🧪 Nucleotide Polymerases

🧬 DNA Polymerases

• Present in all organisms

• Essential for genome replication

• Used in PCR, DNA repair, cloning

🔬 Klenow Fragment (Large Fragment of DNA pol I)

• Derived from E. coli DNA pol I

• Created by removing 5'–3' exonuclease domain

🔧 Activities:

• Polymerase

• 3’–5’ exonuclease (proofreading)

📌 Uses:

• Synthesize dsDNA from ssDNA

• Fill in 3’ overhangs

• Digest 3’ overhangs

• Radioactive probe synthesis

🔥 Thermostable Polymerases (PCR Workhorses)

🔄 Reverse Transcriptase

• Found in retroviruses

• Converts RNA → DNA (cDNA synthesis)

🔧 Activities:

1. RNA-dependent DNA polymerase

2. DNA-dependent DNA polymerase

3. RNase H (removes RNA strand from RNA/DNA hybrid)

🧬 cDNA Synthesis Process:

1. Primer binds RNA

2. DNA strand synthesized (RNA-DNA hybrid)

3. RNA removed → second DNA strand made

✅ Summary Table: DNA Manipulative Enzymes & Cloning