Recombinant DNA (rDNA) technology

Type II (sticky ends)

Type II (blunt ends) Blunt ends are 4-6bp long and pallidromic Means the base sequence in the second half of a DNA strand is the mirror image of the sequence in the first half 5’-GAA AAG-3’ 3’-CTT TTC

S1 nuclease S1 nuclease cuts the single stranded DNA or single strand protrusion of double stranded DNA with cohesive ends These enzymes convert cohesive end into blunt ends E. g Bal31 is very specific single straned endodeoxyriboso nuclease It degrades both 3’and 5’ strands of DNA molecule and shortens the fragments and show blunt ends at both termini

S1 nuclease 5’-AG-3’ S1 nuclease 5’-AG-3’ 3’-TCTTA-5’ 3’-TC-5’ 5’-GGAATT-3’ Bal31 5’GGAA-3’ 3’-CCTTAA-5’ Exonuclease 3’-CCTT-5’

Alkaline phosphatase Enzyme involved in the terminal removal of phosphate group Cohesive ends of plasmids, instead of joining with foreign DNA, join the cohesive ends of the same DNA molecules This problem is overcome by using alkaline phosphatase to digest the terminal 5’phosphonyl group The recombinant DNA has nick with 3’and 5’hydroxyl ends Ligate can joint the both ends of hybrid DNA

E.coli and T4 DNA encode the DNA ligase Seals the single stranded nicks between adjacent nucleotides in a duplex DNA chain The reaction catalysed by these ligases and similar in the host cell but differ in their cofactor requirement T4 needs ATP and E.coli NAD+ Both the enzyme catalyze the formation of phosphodiester bond In each case the cofactor is split into AMP compex and join nick covalently in phosphodiester chain

Cloning Vectors A vector is used to amplify a single molecule of DNA into many copies. A DNA fragment must be inserted into a cloning vector. A cloning vector is a DNA molecule that has an origin of replication and is capable of replicating in a bacterial cell. Any extra chromosomal small genome is used as vector e.g plasmid, cosmid vectors, bacterial artificial chromosomes, and yeast artificial chromosomes.

Plasmid Cloning Vectors Plasmids are circular, double-stranded DNA molecules that exist in bacteria and in the nuclei of some eukaryotic cells. They can replicate independently of the host cell. The size of plasmids ranges from a few kb to near 100 kb Can hold up to 10 kb fragments Plasmids have an origin of replication, antibiotic resistance genes as markers, and several unique restriction sites.

pBR322 : It is 4.6kb double stranded cloning vector developed by Bolivar and Rodriguez

pUC vectors

Cosmid Cloning Vectors COS+ plasmid = cosmid, hybrid vectors derived from plasmids which contain cos site of phase lambda Have unique restriction site origin of replication and selectable markers from the plasmid

Cosmid Cloning Vectors Fragments from 30 to 46 kb can be accommodated by a cosmid vector. Cosmids combine essential elements of a plasmid and Lambda systems. Cosmids are extracted from bacteria and mixed with restriction endonucleases. Cleaved cosmids are mixed with foreign DNA that has been cleaved with the same endonuclease. Recombinant cosmids are packaged into lambda caspids Recombinant cosmid is injected into the bacterial cell where the rcosmid arranges into a circle and replicates as a plasmid.

Shuttle vectors Capable of replicating in two or more types of hosts. Replicate autonomously, or integrate into the host genome and replicate when the host replicates. Possess two ori (oriE orieuk), ampR, ars (autonomous origin of replication), cen (centromere of yeast) and leu-2 gene encoding for leucin Commonly used for transporting genes from one organism to another (i.e., transforming animal and plant cells).

Yeast Artificial Chromosomes(YACs) YACs can hold up to 500 kbs. YACs contain: a yeast centromere, two yeast telomeres, a bacterial origin of replication, and bacterial selectable markers. DNA is inserted to a unique restriction site, and cleaves the plasmid with another restriction endonuclease that removes a fragment of DNA and causes the YAC to become linear. Once in the cell, the rYAC replicates as a chromosome, also replicating the foreign DNA.