Recombinant DNA
RECOMBINANT DNA..................................................................................................................................... 1
GENE CLONING............................................................................................................................................. 2
WHAT IS GENE CLONING.........................................................................................................................................2
GENE CLONING REQUIRES DNA MOLS TO BE CUT IN VERY PRECISE AND REPRODUCIBLE FASHION...........................................2
FUNCTIONS OF CLONING.........................................................................................................................................2
DNA PURIFICATION....................................................................................................................................... 3
TYPES OF DNA NEEDED:.........................................................................................................................................3
1. PREPARATION OF A CELL EXTRACT:........................................................................................................................3
2. REMOVAL OF PROTEIN CONTAMINANTS BY PHENOL EXTRACTION:................................................................................3
3. CONCENTRATION OF DNA:.................................................................................................................................3
Nucleic Acid solubility:....................................................................................................................................4
Role of salt:.....................................................................................................................................................4
Role of Ethanol:..............................................................................................................................................4
Wash step:......................................................................................................................................................4
4. PLASMID PURIFICATION BY ALKALINE DENATURATION................................................................................................4
RESTRICTION ENZYMES................................................................................................................................. 4
BLUNT ENDS.........................................................................................................................................................4
COHESIVE (STICKY) ENDS.........................................................................................................................................5
FREQUENCY OF RECOGNITION SEQUENCES..................................................................................................................5
GEL ELECTROPHORESIS...........................................................................................................................................5
RECOMBINANT MOLECULES.......................................................................................................................... 5
JOINING DNA MOLECULES:.....................................................................................................................................5
TRANSFORMATION.................................................................................................................................................7
Procedure:......................................................................................................................................................7
Efficiency:........................................................................................................................................................7
ISOLATING RECOMBINANT MOLECULES......................................................................................................................7
Insertional Inactivation:..................................................................................................................................7
PLASMID VECTORS........................................................................................................................................ 8
PROPERTIES OF AN IDEAL PLASMID VECTOR.................................................................................................................8
PBR322 – CLASSIC CLONING VECTOR........................................................................................................................8
PUC PLASMIDS.....................................................................................................................................................8
Identification of recombinant PUC.................................................................................................................9
GENE IDENTIFICATION.................................................................................................................................. 9
DIRECT SELECTION...............................................................................................................................................10
Antibiotic Resistance:...................................................................................................................................11
Marker rescue:..............................................................................................................................................11
GENE BANK SCREENING........................................................................................................................................11
Hybridization Probing:..................................................................................................................................12
2 possible ways to generate a DNA probe:..............................................................................................................12
Detection of a cloned gene product.............................................................................................................13
1
, Recombinant DNA
Gene Cloning
What is gene cloning
- A fragment of genomic DNA that includes the gene to be cloned is inserted into a
vector (circular DNA mol). This is then called a recombinant DNA molecule.
- The vector transports the gene into a host cell (usually bacteria)
- Inside host cell vector multiplies, producing numerous identical copies, not only of
itself but also of the gene it carries.
- Host cell divides, copies of the recombinant DNA molecule are passed to the progeny
and further vector replication takes place
- After lots of cell divisions, a colony (or clone) of identical host cells is produced
o Each cell in colony carries >=1 copy of recombinant DNA mol
o Colonies consist of 109 cells.
- Why is it important?
o The technique can provide a pure sample of an individual gene, separated
from all the other genes in the cell
o Each recombinant DNA mol has 1 fragment of genomic DNA, usually each
recombinant DNA mol is transported into a single host cell, so each colony
contains cells with multiple copies of just one gene fragment
- The key to the success of cloning is the ability to identify the particular clone of
interest from many different clones that are obtained.
Gene cloning requires DNA mols to be cut in very
precise and reproducible fashion
- Each vector mol must be cleaved at a single position to open up circle so new DNA
can be inserted
- A mol cut >1 times will be broken into separate fragments and will be of no use as a
cloning vector
- Each vector mol must be cut at exactly the same position on the circle
- Genomic DNA must also be cleaved for 2 reasons:
o If aim is to clone a single gene (2 or 3 kb of DNA) then that gene will have to
be cut out of the large (>80kb) DNA mols
o Large DNA mols will need to be cut simply to produce fragments small
enough to be carried by the vector.
Most cloning vectors exhibit a preference for DNA fragments that fall
into a particular size range
Functions of cloning
1. Production of large numbers of rDNA (recombinant DNA) molecules from a limited
quantity of starting material
a. Few ng DNA -> replicates in host cell form 15-1000 copies per cell -> cell
replicates to form a colony with 7ug per colony (1000x increase) -> colony
inoculated in 500ml liquid broth forms 7 mg rDNA (million-fold increase)
2. Purification of desired recombinant DNA fragment
2
RECOMBINANT DNA..................................................................................................................................... 1
GENE CLONING............................................................................................................................................. 2
WHAT IS GENE CLONING.........................................................................................................................................2
GENE CLONING REQUIRES DNA MOLS TO BE CUT IN VERY PRECISE AND REPRODUCIBLE FASHION...........................................2
FUNCTIONS OF CLONING.........................................................................................................................................2
DNA PURIFICATION....................................................................................................................................... 3
TYPES OF DNA NEEDED:.........................................................................................................................................3
1. PREPARATION OF A CELL EXTRACT:........................................................................................................................3
2. REMOVAL OF PROTEIN CONTAMINANTS BY PHENOL EXTRACTION:................................................................................3
3. CONCENTRATION OF DNA:.................................................................................................................................3
Nucleic Acid solubility:....................................................................................................................................4
Role of salt:.....................................................................................................................................................4
Role of Ethanol:..............................................................................................................................................4
Wash step:......................................................................................................................................................4
4. PLASMID PURIFICATION BY ALKALINE DENATURATION................................................................................................4
RESTRICTION ENZYMES................................................................................................................................. 4
BLUNT ENDS.........................................................................................................................................................4
COHESIVE (STICKY) ENDS.........................................................................................................................................5
FREQUENCY OF RECOGNITION SEQUENCES..................................................................................................................5
GEL ELECTROPHORESIS...........................................................................................................................................5
RECOMBINANT MOLECULES.......................................................................................................................... 5
JOINING DNA MOLECULES:.....................................................................................................................................5
TRANSFORMATION.................................................................................................................................................7
Procedure:......................................................................................................................................................7
Efficiency:........................................................................................................................................................7
ISOLATING RECOMBINANT MOLECULES......................................................................................................................7
Insertional Inactivation:..................................................................................................................................7
PLASMID VECTORS........................................................................................................................................ 8
PROPERTIES OF AN IDEAL PLASMID VECTOR.................................................................................................................8
PBR322 – CLASSIC CLONING VECTOR........................................................................................................................8
PUC PLASMIDS.....................................................................................................................................................8
Identification of recombinant PUC.................................................................................................................9
GENE IDENTIFICATION.................................................................................................................................. 9
DIRECT SELECTION...............................................................................................................................................10
Antibiotic Resistance:...................................................................................................................................11
Marker rescue:..............................................................................................................................................11
GENE BANK SCREENING........................................................................................................................................11
Hybridization Probing:..................................................................................................................................12
2 possible ways to generate a DNA probe:..............................................................................................................12
Detection of a cloned gene product.............................................................................................................13
1
, Recombinant DNA
Gene Cloning
What is gene cloning
- A fragment of genomic DNA that includes the gene to be cloned is inserted into a
vector (circular DNA mol). This is then called a recombinant DNA molecule.
- The vector transports the gene into a host cell (usually bacteria)
- Inside host cell vector multiplies, producing numerous identical copies, not only of
itself but also of the gene it carries.
- Host cell divides, copies of the recombinant DNA molecule are passed to the progeny
and further vector replication takes place
- After lots of cell divisions, a colony (or clone) of identical host cells is produced
o Each cell in colony carries >=1 copy of recombinant DNA mol
o Colonies consist of 109 cells.
- Why is it important?
o The technique can provide a pure sample of an individual gene, separated
from all the other genes in the cell
o Each recombinant DNA mol has 1 fragment of genomic DNA, usually each
recombinant DNA mol is transported into a single host cell, so each colony
contains cells with multiple copies of just one gene fragment
- The key to the success of cloning is the ability to identify the particular clone of
interest from many different clones that are obtained.
Gene cloning requires DNA mols to be cut in very
precise and reproducible fashion
- Each vector mol must be cleaved at a single position to open up circle so new DNA
can be inserted
- A mol cut >1 times will be broken into separate fragments and will be of no use as a
cloning vector
- Each vector mol must be cut at exactly the same position on the circle
- Genomic DNA must also be cleaved for 2 reasons:
o If aim is to clone a single gene (2 or 3 kb of DNA) then that gene will have to
be cut out of the large (>80kb) DNA mols
o Large DNA mols will need to be cut simply to produce fragments small
enough to be carried by the vector.
Most cloning vectors exhibit a preference for DNA fragments that fall
into a particular size range
Functions of cloning
1. Production of large numbers of rDNA (recombinant DNA) molecules from a limited
quantity of starting material
a. Few ng DNA -> replicates in host cell form 15-1000 copies per cell -> cell
replicates to form a colony with 7ug per colony (1000x increase) -> colony
inoculated in 500ml liquid broth forms 7 mg rDNA (million-fold increase)
2. Purification of desired recombinant DNA fragment
2
