Download What is gene cloning?

What is gene cloning?
Why gene cloning and PCR are so important?
- Gene cloning allows individual fragments of DNA
to be purified.
- Gene cloning allows to study expression and
function of an individual gene.
- Broad application of Gene cloning and PCR
; - biotechnology
- medicine
- agriculture
- forensic science
Part I
The basic principles of gene cloning and DNA analysis
Part II
Applications of gene cloning and DNA analysis in research
Part III
The applications of gene cloning and DNA analysis in biotechnology
Part I
The basic principles of gene cloning and DNA analysis
Why gene cloning and DNA analysis are important?
Vectors for gene cloning: plasmids & bacteriophages
Cloning vectors for E. coli
Cloning vectors for eukaryotes
Purification of DNA from living cells
Manipulation of purified DNA
Introduction of DNA into living cells
How to obtain a clone of a specific gene
The polymerase chain reaction
Part I
The basic principles of gene cloning and DNA analysis
Part II
Applications of gene cloning and DNA analysis in research
Part III
The applications of gene cloning and DNA analysis in biotechnology
Part II
Applications of gene cloning and DNA analysis in research
Sequencing genes and genomes
Studying gene expression and function
Studying genomes
- Genome annotation
- Transcriptome
- Proteome
Part I
The basic principles of gene cloning and DNA analysis
Part II
Applications of gene cloning and DNA analysis in research
Part III
The applications of gene cloning and DNA analysis in biotechnology
Part III
The applications of gene cloning and DNA analysis in biotechnology
Production of protein from cloned genes
Gene cloning and DNA analysis in medicine
Gene cloning and DNA analysis in agriculture
Gene cloning and DNA analysis in forensic science & archaeology
Vectors for gene cloning
- Plasmids in bacteria
- Bacteriophages
Vectors for gene cloning
- must be able to replicate within the host cells.
- need to be less than 10 kb in size
plasmids
- Circular-form DNA (Fig. 2.1)
- Independent existence in the bacterial cells
- Carry antibiotics resistance genes
: ex) ampicillin resistant gene (AmpR)
→ can be used as a selectable marker (Fig. 2.2)
- Possess an origin of replication (Fig. 2.3)
2.1 plasmids
- Circular-form DNA (Fig. 2.1)
- Independent existence in the bacterial cells
- Carry antibiotics resistance genes
: ex) ampicillin resistant gene (AmpR)
→ can be used as a selectable marker (Fig. 2.2)
- Possess an origin of replication (Fig. 2.3)
Replication strategies
2.1.1 Size and copy number
- Size from about 1 kb to above 250 kb (Table 2.1)
- Copy number
: the number of molecules of a plasmid contained in a single cell
* stringent plasmid; 1 or 2 low copy number
* relaxed plasmid; 50 or more per cell - a useful cloning vector
2.1.2 Conjugation and compatibility
Conjugation:
- Physical contact b/w two bacteria, usually
associated with transfer of DNA
- is controlled by tra genes, which are present
on conjugative plasmids.
Compatibility:
- the ability of different types of plasmid to
coexist in the same cell
2.1.3 Plasmid classification
- is based on the main characteristic coded by the plasmid genes.
1. Fertility or F plasmid:
- carry only tra genes → promote conjugal transfer of plasmid
- example: F plasmid of E. coli
2. Resistance or R plasmids:
- carry genes conferring on the host bacterium resistance to antibacterial agents.
(ampicillin, tetracyclin, chloramphenicol, mercury)
3. Col plasmids:
- code for colicins, proteins that kill other bacteria
- example: ColE1 of E. coli
4. Virulence plasmids:
- confer pathogenicity on the host bacterium
- example: Ti plasmid of Agrobacterium tumefaciens
2.1.3 Plasmids in organisms other than bacteria
- Eukaryotic plasmid:
* 2 mM circle of yeast Saccharomyces cerevisiae
- Many higher organisms simply do not harbor plasmids within the cells.
Vectors for gene cloning
- Plasmids in bacteria
- Bacteriophages
2.2 Bacteriophages
-
Viruses that specifically infect bacteria
-
Consisting of DNA or RNA molecules carrying genes coding for replication & capsid
proteins
2.2.1 The phage infection cycle
Lytic cycle: T2, T4, T7 phages
Lytic cycle + lysogenic cycle: l phage
Lysogenic cycle: M13 phage
2.2.1 The phage infection cycle
In lytic infection cycle;
-
Phage DNA replication is immediately
followed by synthesis of capsid proteins.
-
Phage DNA molecules is not maintained in a
stable-form in the host cell.
2.2.1 The phage infection cycle
Lytic cycle: T2, T4, T7 phages
Lytic cycle + lysogenic cycle: l phage
Lysogenic cycle: M13 phage
Lytic versus lysogenic infection by phage l
l repressor protein ↑
prophage
lysogen
Mutagenic chemicals
The role of cos site l DNA molecule
49 kb
1. Circularization of the linear DNA molecule: necessary for insertion into
the bacterial genome.
2. acts as a recognition sequences for an endonuclease that cleaves the catenane at
the cos sites, producing individual l genomes.
3. The packaging processes recognize just the cos sites.
Rolling circle replication (s replication)
serves as a primer (plus strand)
by endonuclease
Cloning with a l insertion (replacement) vector
In vitro packaging & amplification of l recombinant DNA
Inoculation of plaque in E. coli liquid-culture medium and
purification of recombinant plasmid
2.2.1 The phage infection cycle
Lytic cycle: T2, T4, T7 phages
Lytic cycle + lysogenic cycle: l phage
Lysogenic cycle: M13 phage
The lysogenic cycle of bacteriophage M13
Rolling circle replication that produces single-stranded circular progeny DNAs
serves as a primer (plus strand)
endonuclease
Several features of M13 as a cloning vector
- Desirable size for cloning vector (10 kb)
- Double-stranded form in infected E. coli cells
- Single-stranded form in culture medium of
infected E. coli:
* useful for DNA sequencing
* useful for in vitro mutagenesis
* useful for phage display