Once recombinant plasmid is constructed, it is introduced into recipient cells. Introduction of recombinant DNA into recipient cells is called transformation: introduction of foreign DNA changes (transforms) properties of the organism.
Special treatment makes cells competent - capable of accepting foreign DNA. Usually, these treatments make cell membrane more permeable for a DNA molecule. When competent cells are mixed with DNA some cells (actually, very few) become transformed: they acquire recombinant vector DNA.
After transformation, cells are plated onto agar medium that contains selective antibiotic: only transformed cells, that acquire antibiotic resistance gene on the vector plasmid, will survive and form colonies. All the untransformed cells will die.
In each colony formed on the agar plate, all cells are descendants of one transformed cell.
All cells in the clone are genetically identical and contain the same recombinant vector
Summarizing:
A typical gene-cloning experiment includes:
CHOOSING A CLONING VECTOR
INTRODUCING FOREIGN DNA INTO THE VECTOR
TRANSFORMATION OF COMPETENT CELLS
SELECTION OF TRANSFORMED CELLS
GENE LIBRARIES
How to clone one particular gene from thousands of genes present in the genome? One of the most frequently used way to clone a specific gene is to use a GENE LIBRARY approach:
Clone everything you can and then find what you need.
How to clone a specific gene?
After plating on agar plates containing the selective antibiotic,only cells with plasmids will survive. Plating makes possible to screen out cells that did not get any plasmid.
Each transformed cell that acquired a plasmid forms a colony on selective medium. All cells in a colony have the same plasmid. Cells in different colonies have different plasmids.
How to find the clone with "our" gene?
The most common methods include:
1. Phenotypic screening
2. Screening with antibodies
3. DNA hybridization
Phenotypic screening is used when cloned gene is expressed and changes properties of the cell in an "obvious way". In the shown example, the protein encoded in the cloned gene changes the color of transformed cells.
Screening with antibodies is used when cloned gene is expressed and antibodies recognizing the encoded protein are available.
DNA hybridization
If the nucleotide sequence of
the whole gene, or any part of it, is known, then DNA hybridization
can be used to find the clone in the library that contains the
cloned gene.
Only one colony on this plate contains DNA with the nucleotide sequence of the gene we are looking for
Cloning euykaryotic genes in prkaryotes require special "tricks" because eukaryotic genes have introns which are removed in teh nucleus of eukaryotic cells prior to translation
Introns can account for more than 90% of the length of a eukaryotic gene. It is hard to clone very long DNA segments. In addition, intron-containing eukaryotic genes cannot be expressed in a bacterial host because prokaryotes lack splicing apparatus. To overcome these problems, instead of directly cloning a gene, one can clone cDNA, a DNA copy of gene mRNA.
An enzyme, reverse transcriptase, is used to produce cDNA
In construction of cDNA libraries we make use of the fact that all translated mRNAs in eukaryotic cells contain poly (A) tail.
An oligonucleotide with the sequence
TTTTTTTTT can be hybridized to the poly (A) tail of fully processed
mRNAs and used as a primer for synthesis of cDNA (a DNA strand complementary to mRNA)
cDNA can be introduced into a cloning vector and cloned in a bacterial host in order to study gene structure or to express gene product