Transcript Lecture# 2 Recombinant DNA technology - UG 2014

Lecture# 2
Recombinant DNA technology
Dr. Shah Rukh Abbas
ASAB, NUST.
I. DNA is the genetic material of most
organisms (from bacteria to humans)
Plasmid
Chromosome: Most bacteria have one circular DNA chromosome ranging in size from
1,000 to 8,000 kilo base pairs.
Plasmid: Extra chromosomal genetic element also made of a circular DNA molecule.
Bacterial Genome: The collection of all of the genes present on the bacteria’s
chromosome or its extra chromosomal genetic elements.
Basics: Nucleotides are the building
blocks of DNA
Chemical structure of DNA & RNA
Chemical structure of dsDNA
Chemical structure of dsDNA
II. Restriction Endonucleases (RE) enzymes
• Enzymes that act as scissors to cut the DNA at specific sites.
– 1962: “molecular scissors” discovered in bacteria.
• E. coli bacteria have an enzymatic immune system that
recognizes and destroys foreign DNA’s sugar phosphate back
bone.
• Restriction Endonucleases recognize specific sequence with
the DNA molecules
• The recognized sequence are usually four to six base pair and
are palindromic.
Bam H1 site:
5’-GGATCC-3’
3’-CCTAGG-5’
• Named for bacterial genus, species, strain, and type. Example:
EcoR1
Genus: Escherichia, Species: coli Strain: R Order discovered: 1
• Types of RE enzymes
• Type I enzymes are complex, multisubunit, combination
restriction-and-modification enzymes that cut DNA at random
far from their recognition sequences.
• Type II Type II enzymes cut DNA at defined positions close to
or within their recognition sequences. They produce discrete
restriction fragments and distinct gel banding patterns, and
they are the only class used in the laboratory for routine DNA
analysis and gene cloning.
• Type III enzymes cleave DNA from 24 to 26 bp away from the
recognition site.
• Type IV enzymes recognize modified, typically methylated
DNA and are exemplified by the McrBC and Mrr systems of E.
coli.
Restriction Enzymes
Restriction Enzymes Cutting Sites
Blunt Ends
AluI
HaeIII
Sticky Ends
HindIII
EcoRI
Why don’t bacteria destroy their own DNA with their
restriction enzymes?
What are exonucleases?
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end
(exo) of a polynucleotide chain. A hydrolyzingreaction that breaks phosphodiester bonds
at either the 3’ or the 5’ end occurs.
http://highered.mheducation.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0
072437316/120078/bio37.swf::Restriction+Endonucleases
Uses for Restriction Enzymes
RFLP analysis (Restriction Fragment Length Polymorphism)
DNA sequencing
DNA storage – libraries
Transformation
Large scale analysis – gene chips
Restriction Enzymes for RFLP
_
DNA is negatively
charged from the
phosphate backbone
+
Visualize DNA with ethidium
bromide – fluoresces ONLY
when bound to DNA
III. Pasting DNA using DNA ligase
• Complementary
ends (sticky ends)
H-bond
• Ligase forms
phosphodiester
bond to seal
strands together.
III. Vectors: Bacterial Plasmids
Allowing the exogenous DNA to be inserted, stored, and manipulated
mainly at DNA level.
TYPES:
1 Plasmid vectors
2 Bacteriophage vectors
3 Cosmids
4 BACs & YACs
Must have:
• Ori of replication and in case of expression vector
gene expression mechanism (operon etc).
• Marker- to indicate recombinant
• Multiple cloning site
http://www.dnai.org/b/index.html
1. Plasmids
• Plasmids - circular DNA duplexes of 1 to 200 kb that contain the
requisite genetic machinery (replication origin) necessary for
autonomous replication in bacteria or yeast.
• Types of plasmids are determined by their copy number.
• Stringent control- one to a few copies per cell.
• Relaxed control- 10 to 700 copies per cell-if protein synthesis is
inhibited by an antibiotic (chloramphenicol), the plasmid will
continue to replicate up to 2000-3000 copies.
• Plasmids used in molecular cloning are
– relatively small
– replicate under relaxed control
– carry genes for antibiotic resistance
– number of restriction sites (polylinker) for inserting DNA
segments.
• Cannot be used to clone DNAs longer than ~10 kb.
• Blue-white screeing.
1. Plasmids
• Plasmid vectors are ≈1.2–3kb and
contain:
– Replication origin (ORI)
sequence
– A gene that permits selection,
Here the selective gene is ampr; it
encodes the enzyme b-lactamase,
which inactivates ampicillin.
– Exogenous DNA can be inserted
into the bracketed region .
Selective Marker
• Selective marker is required for
maintenance of plasmid in the cell.
• Because of the presence of the
selective marker the plasmid
becomes useful for the cell.
• Under the selective conditions, only
cells that contain plasmids with
selectable marker can survive
• Genes that confer resistance to
various antibiotics are used.
• Genes that make cells resistant to
ampicillin, neomycin, or
chloramphenicol are used
Origin of Replication
• Origin of replication is a
DNA segment recognized
by the cellular DNAreplication enzymes.
• Without replication origin,
DNA cannot be replicated
in the cell.
Multiple Cloning Sites
• Many cloning vectors contain a
multiple cloning site or polylinker:
a DNA segment with several unique
sites for restriction endo- nucleases
located next to each other
• Restriction sites of the polylinker
are not present anywhere else in
the plasmid.
• Cutting plasmids with one of the
restriction enzymes that recognize a
site in the polylinker does not
disrupt any of the essential features
of the vector
Plasmid vectors
• Advantages:
– Small, easy to handle
– Straightforward selection strategies
– Useful for cloning small DNA fragments
(< 10kbp)
• Disadvantages:
– Less useful for cloning large DNA fragments
(> 10kbp)
2. Bacteriophage vectors
• Advantages:
– Useful for cloning large DNA fragments
(10 - 23 kbp)
– Inherent size selection for large inserts
• Disadvantages:
– Less easy to handle
l vectors
• Left arm:
– head & tail proteins
• Right arm:
– DNA synthesis
– regulation
– host lysis
• Deleted central region:
– integration & excision
– regulation
l Vector (M-13
3. Cosmid vectors
Combine the properties of plasmid vectors
with the useful properties of the l cos site
• Advantages:
– Useful for cloning very large DNA fragments
(32 - 47 kbp)
– Inherent size selection for large inserts
– Handle like plasmids
• Disadvantages:
– Not easy to handle very large plasmids
– (~ 50 kbp)
l ZAP
4. BACs and YACs
BACs : Bacterial Artificial Chromosomes
YACs : Yeast Artificial Chromosomes
• Advantages:
– Useful for cloning extremely large DNA fragments
(100 - 2,000 kbp)
– This is very important for genome sequencing
projects
• Disadvantages:
– Not easy to handle extremely large DNA
molecules
BAC vector
• oriS and oriE
mediate replication
• parA and parB
maintain single
copy number
• ChloramphenicolR
marker
YAC vector
large
inserts
ARS
URA3
telomere
centromere
replication
origin
HIS3
markers
telomere
• Capable of carrying inserts of 200 - 2000 kbp in yeast