Timeline / Sequence: Steps in rDNA Technology

Timeline of rDNA Technology Development

Year	Event	Significance
1952	Restriction-modification system discovered (Luria and Human)	First evidence of bacterial restriction of phage
1970	First restriction enzyme (HindII) isolated by Hamilton Smith	Proved sequence-specific DNA cutting
1972	Paul Berg creates first recombinant DNA molecule (SV40 + lambda DNA)	Birth of genetic engineering
1973	Cohen and Boyer: first recombinant DNA cloned in E. coli	Practical foundation of biotechnology
1977	pBR322 constructed by Bolivar and Rodriguez	Model cloning vector for lab and teaching
1978	Nobel Prize: Arber, Nathans, Smith for restriction enzymes	Recognition of foundational work
1982	First recombinant human insulin (Humulin) approved by FDA	First commercial biotech therapeutic
1983	Kary Mullis invents PCR	Revolutionary amplification technique
1988	Taq polymerase replaces Klenow in PCR	PCR becomes automatable in thermal cyclers
1993	Kary Mullis receives Nobel Prize in Chemistry for PCR	Global recognition of PCR's impact
1996	Dolly the sheep cloned by somatic cell nuclear transfer	Applications of genetic manipulation
2003	Human Genome Project completed	PCR-based sequencing of entire human genome

Step-by-Step Sequence: Complete rDNA Cloning

1. ISOLATE target gene
   └── From genomic DNA (if prokaryotic) OR cDNA (if eukaryotic gene with introns)

2. CUT with restriction enzyme
   └── Same enzyme for insert AND vector → complementary sticky ends

3. MIX insert + vector
   └── Sticky ends hybridize by complementary base pairing

4. LIGATE with DNA ligase
   └── Phosphodiester bonds seal the nicks → recombinant plasmid formed

5. TRANSFORM into competent host
   └── CaCl2 + heat shock (42°C) OR electroporation OR biolistics

6. SELECT transformed cells
   └── Plate on ampicillin medium → only cells with plasmid survive

7. SCREEN for recombinants
   └── Replica plate on tetracycline → grows on Amp not Tet = recombinant

8. CULTURE recombinant clones
   └── In bioreactor for scale-up

9. DOWNSTREAM PROCESS
   └── Separation → Purification → Formulation → QC → Final product