Comparison Note: Restriction Enzymes, DNA Ligase, and Vectors

Property	Restriction Enzyme (EcoRI)	DNA Ligase (T4)	pBR322 Vector	Ti Plasmid
Source	Escherichia coli RY13	Bacteriophage T4	Artificial (from E. coli plasmids)	Agrobacterium tumefaciens
Function	Cuts DNA at GAATTC; produces sticky ends	Joins DNA fragments; seals phosphodiester bonds	Carries foreign DNA; replicates in E. coli	Transfers T-DNA into plant chromosomes
End type	Sticky (5'-AATT overhang)	N/A (joining, not cutting)	Has restriction sites for cloning	T-DNA integrates into plant genome
Hosts	Bacteria (E. coli)	In vitro or any cell	E. coli (bacteria only)	Dicot plants (not monocots efficiently)
Key feature	Palindrome recognition; >900 known enzymes	Energy from ATP (T4) or NAD+ (bacterial)	Has ampR, tetR, ori; 4361 bp	Disarmed (tumor genes removed); T-DNA retained
NEET focus	Recognition sequence, end type	Molecular glue; completes cloning	Insertional inactivation	Plant transformation; not for monocots

Method	Organism Used For	Mechanism	Advantage	Limitation
CaCl2 + heat shock	Bacteria (E. coli)	CaCl2 neutralizes charges; 42°C heat shock	Simple, cheap	Only for E. coli; low efficiency
Electroporation	Bacteria, yeast, mammalian	Brief electrical pulses → transient membrane pores	High efficiency; versatile	Requires equipment; may damage cells
Microinjection	Animal cells, oocytes	Glass needle injects DNA into nucleus	Very precise	Very low throughput
Biolistics (gene gun)	Plant cells, organelles	DNA-coated Au/W particles fired at high velocity	Works on plant walls; transforms organelles	Requires equipment; variable efficiency