Transcript Recombinant DNA Technology (d)

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’
3’-TCTTA-5’
S1 nuclease 5’-AG-3’
3’-TC-5’
5’-GGAATT-3’ Bal31
3’-CCTTAA-5’ Exonuclease
5’GGAA-3’
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
1. Capable of replicating in two or more types of hosts.
2. Replicate autonomously, or integrate into the host genome and
replicate when the host replicates.
3. Possess two ori (oriE orieuk), ampR, ars (autonomous origin of
replication), cen (centromere of yeast) and leu-2 gene encoding for
leucin
4. 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.