Chapter 8.0 RECOMBINANT DNA TECHNOLOGY · Cloning Vectors: Cosmid • Plasmid with yeast chromosome...
Transcript of Chapter 8.0 RECOMBINANT DNA TECHNOLOGY · Cloning Vectors: Cosmid • Plasmid with yeast chromosome...
Chapter 8.0
RECOMBINANT DNA TECHNOLOGY
OVERVIEW
RECOMBINANT
DNA
TECHNOLOGY
METHODS
IN GENE
CLONING
APPLICATION OF
RECOMBINANT DNA
TECHNOLOGY
• Define recombinant DNA technology.
b) Define and explain the tools used in recombinant DNA technology, target DNA, restriction enzymes, DNA cloning vector, host cell and modifying enzymes.
(c)Explain restriction enzyme and examples of enzymes that produce sticky ends.
• (EcoRI: G/AATTC) and blunt ends (SmaI : CCC/GGG)
(c)Explain the characteristics of plasmid as cloning and expression vector
(d)Explain the characteristic of E.coli as host cell and its characteristics
(e)Explain modifying enzyme and its function; (i) DNA ligase for DNA ligation
• (ii) Taq polymerase for DNA amplification using PCR.
Learning
Outcomes
Definition of Recombinant DNA Technology
• Formation of new combinations of genes by
isolating genes from the organism and introducing
them into either a similar or unrelated organism.
PURPOSES Enable scientists to obtain many copies of specific DNA segment for the purpose of
studying it
Modifying the DNA of an organism to produce new
genes with new traits.
Tools Used
in Cloning
Target DNA
Host Cell
Modifying
Enzymes Restriction
Enzyme
DNA Cloning Vector
Target DNA
A selected DNA that contain gene of interest.
Restriction enzymes
A molecular scissors that cut the single strand
and the double strand at specific point
Examples: EcoR1 to produce sticky ends Sma1 to produce blunt ends
DNA CLONING VECTOR
A plasmid of bacteria that brings the foreign DNA fragment
into the genome of the host cell
Examples: Plasmid, Bacteriphage, Cosmid, Yeast Artificial Chromosomes
HOST CELL
A cell that receives recombinant DNA
for cloning purpose
HOST CELL
Enzymes used in join targeted DNA fragment with
the vector to form recombinant DNA.
MODIFYING ENZYME
Example: DNA Ligase, Taq polymerase
Restriction endonucleases.
• Extracted from bacteria.
• Naturally used to cut viral DNA into small
fragments at specific base/site.
– For defense.
Restriction Enzymes
RESTRICTION ENZYME
Enzymes Source
EcoRI Escherichia coli
BamHI Bacillus amyloliquefaciens
SmaI Serratia marcescens
• Splice through DNA at specific base sequence.
• Specific base sequence: Restriction sites.
• Most of the base are palindromic.
Restriction sites
• Palindromic?
Base sequence of one strand reads the same as its complement strand in opposite direction.
Enzymes Restriction Sites
EcoRI 5’-GAATTC-3’ 3’-CTTAAG-5’
BamHI 5’-GGATCC-3’ 3’-CCTAGG-5’
SmaI 5’-GGGCCC-3’ 3’-CCCGGG-5’
Example: EcoRI
• Most make staggered cut in two strands forming sticky ends.
breaking the phosphodiester bond
RESTRICTION ENZYME THAT PRODUCE STICKY ENDS:
• Example: SmaI
• Some cut straight across both strand forming blunt ends.
RESTRICTION ENZYME THAT PRODUCE BLUNT ENDS:
DNA cloning vector
• A small piece of DNA which a foreign DNA fragment can be inserted.
• Example: Plasmid
Bacteriophage
Cosmid
Yeast artificial chromosomes (YACs)
Types
DNA Inserts Capacity
(1 kb = 1000 nucleotides)
Form of vector Example
Plasmid 10 kb Double stranded
circular DNA pUC18
Bacteriophage 20 kb Linear DNA λ2001
Cosmid 35-45 kb Double stranded
circular DNA sCOS-1
YACs – Yeast Artificial Chromosomes
200-1500 kb Double stranded
circular DNA
pYAC
Cloning Vectors: Plasmid
21
• Small ring-shape DNA
in bacteria.
• Not part of
chromosome.
• Self-replicating.
• Has small number of
genes.
• Example: pUC18
Cloning
• Viruses that infect bacteria
• Can carry larger DNA
inserts than bacterial plasmid
• Eg. λ2001
Cloning Vectors: Bacteriophage
• Hybrids of plasmid
and bacteriophage lambda DNA.
• Can accommodate large inserts of DNA
• eg.: Scos-1
Cloning Vectors: Cosmid
• Plasmid with yeast chromosome
• Eg. pYAC – capacity
is bigger than other vectors
Cloning Vectors: YACs
Cloning vector: Characteristics
1. Able to accept
foreign DNA in
multiple cloning sites (MCS).
Example: Plasmid
Multiple Cloning
Sites
2. Able to replicate freely in host cell.
Present of origin of
replication initiation -ori gene
Cloning vector: Characteristics
3.Possess selectable genetic marker
a. resistance to antibiotic
eg: ampR, tetR,
kanR
b. lacZ gene
encode for β-
galactosidase
Cloning vector: Characteristics
Host Cell
A cell that can be utilized for DNA cloning in order
to accept, maintain and allow the reproduction of
cloning vector.
Example: Bacteria
1. Able to receive DNA recombinant through the transformation process.
Host cell: Characteristics
2. Able to maintain the structure of DNA recombinant from one generation to other.
Host cell: Characteristics
3. Able to amplify the gene product from the DNA recombinant.
gene product e.g insulin
Host cell: Characteristics
Modifying Enzymes
• Enzymes used in modification of DNA.
Example: DNA Ligase
✓Catalyzed the formation of phosphodiester bonds between adjacent nucleotides in DNA.
• Modifying enzymes: Joins targeted DNA fragment with the vector to form recombinant DNA.
• Examples of modifying enzymes are: DNA ligase and Taq polymerase
NEXT LECTURE
METHODS IN GENE CLONING