Cloning Vectors & Hosts
โญ Vector Insert Capacity Core
5 Parameters
Cloning Vectors: Plasmids vs Bacteriophage Lambda vs Cosmids vs BACs vs YACs
Comparative matrix of insert capacity, host organisms, origin of replication, and genomic library suitability.
| Vector Class | Insert Size Capacity | Host Organism | Key Structural Element & Feature |
|---|---|---|---|
| Plasmid (e.g. pBR322, pUC19) | 0.5 โ 10 kb (efficiency drops drastically >10 kb) | Escherichia coli | ColE1 origin, antibiotic selectable markers (AmpR, TetR), MCS in lacZ' |
| Bacteriophage Lambda (ฮป) | 9 โ 23 kb (replacement vectors like ฮป-EMBL4) | Escherichia coli | In vitro packaging into phage heads via cos sites; high transfection yield |
| Cosmid (Plasmid + cos sites) | 30 โ 45 kb | Escherichia coli | Plasmid containing phage ฮป cos sites; packaged as phage, replicates as plasmid |
| Bacterial Artificial Chromosome (BAC) | 100 โ 300 kb | Escherichia coli | Based on E. coli F-factor plasmid (oriS, repE); maintained at stable 1โ2 copies/cell |
| Yeast Artificial Chromosome (YAC) | 100 โ 1000+ kb (1 Mb; Largest capacity) | Saccharomyces cerevisiae | Linear chromosome with yeast ARS (origin), CEN (centromere), and TEL (telomeres) |
Insert Capacity Hierarchy
Key Point
Vector: Plasmid
Max Insert: 10 kb
Host: E. coli
Key Point
Vector: Phage ฮป
Max Insert: 23 kb
Host: E. coli
Key Point
Vector: Cosmid
Max Insert: 45 kb
Host: E. coli
Key Point
Vector: BAC
Max Insert: 300 kb
Host: E. coli
Key Point
Vector: YAC
Max Insert: 1,000 kb (1 Mb)
Host: Yeast
Restriction Endonucleases
โญ Molecular Scissors Benchmark
5 Parameters
Restriction Endonucleases: Type I vs Type II vs Type III
Comparative matrix of cleavage site proximity, cofactor requirements, bifunctional activity, and recombinant DNA utility.
| Property | Type I Restriction Enzyme | Type II (Recombinant Standard) | Type III Restriction Enzyme |
|---|---|---|---|
| Cleavage Site Relative to Recognition Site | Cleaves non-specifically at variable distance >1000 bp away from recognition site | Cleaves SPECIFICALLY AT or within adjacent 4โ8 bp palindromic recognition sequence | Cleaves specific site ~24 โ 26 bp downstream of recognition sequence |
| Enzymatic Architecture | Multimeric bifunctional enzyme (restriction and methylase in same complex) | Separate and distinct restriction endonuclease and methylase enzymes | Heterodimeric enzyme sharing subunits |
| Cofactors Required for Cleavage | ATP, S-adenosylmethionine (SAM), and Mg2+ | Mg2+ ONLY (No ATP or SAM needed) | ATP and Mg2+ (SAM stimulates but not essential) |
| Recombinant DNA (rDNA) Utility | NONE (random cleavage creates heterogeneous uncontrollable fragments) | INDISPENSABLE (predictable, reproducible cleavage with sticky or blunt ends) | Very limited; rarely used in molecular cloning |
| Classic Examples | EcoK, EcoB | EcoRI, HindIII, BamHI, PstI, NotI | EcoP1, EcoP15 |
Cleavage Specificity & Cofactors
Type I
Cleavage Position: >1000 bp away (Random)
Cofactors: ATP + SAM + Mg2+
Cloning Use: None
Type II
Cleavage Position: AT Recognition Site
Cofactors: Mg2+ only
Cloning Use: STANDARD (Universal)
Type III
Cleavage Position: 25 bp downstream
Cofactors: ATP + Mg2+
Cloning Use: None
