GENE CLONING

The term clone was derived from Greek word it mean twig or branch. Clone is exact copy of an organism, single cell, organelle or macromolecule. The first step in cloning is isolation of one of the few to thousands of genes present in the cells of organisms. For this purpose to detect particular gene is called Shot Gun method.

A tool kit for recombinant DNA technology

1. Tools to cut DNA molecules into large gene-sized fragments 2. Vector DNA molecules that can replicate and into which foreign DNA can be inserted 3. Tools to join vector DNA and foreign DNA together to create recombinant DNA molecules 4. Methods for introducing recombinant DNA molecules into bacterial cells where they can replicate 5. A method for identifying bacterial cells that have taken up the recombinant DNA molecules = a method of selecting for transformants 1. Tools for cutting DNA molecules into gene-sized fragments a. b. genes c. d. e. f. A typical chromosome might contain more than 5 million base pairs Molecules of this enormous size cannot easily be cloned and they contain 1000s of A single gene might contain approximately 2,000 base pairs of DNA sequence. Molecules of this size can be easily cloned For this reason, we need to be able to cut the very large chromosome-sized DNA molecules into smaller gene-sized fragments This can be done using a cutting tool called a restriction enzyme

Restriction enzymes

Restriction enzymes are made by bacteria to protect themselves incoming virus DNA They act as molecular scissors to cut DNA into large fragments They have a unique property: they cut DNA in a sequence-specific manner by recognizing a specific sequence of bases e.g. The restriction enzyme EcoRI will only cut a DNA molecule if it contains the following sequence: ---------G-A-A-T-T-C----------------C-T-T-A-A-C--------

Restriction enzymes cut DNA infrequently

The frequency with which this particular sequence of 6 base pairs will occur in a very large DNA molecule can be calculated on the basis of: the probability of occurrence of any specified base at each position: Because this enzymes cuts DNA only once in every 4,000 base pairs (on average), the fragments are sufficiently large to contain an entire gene

2. DNA Cloning Vectors

a. A cloning vector is a DNA molecule that can contains information for replicating itself a so-called origin of replication b. If a piece of foreign DNA is incorporated into the vector, then the foreign DNA will be replicated along with the vector DNA c. The most commonly used type of cloning vector is a small (4000 bp) circular DNA molecule called a plasmid d. Plasmids are found naturally in various types of bacteria e. Plasmids can replicate independently of the bacterial chromosome because they have an origin of replication

3. Making recombinant DNA molecules

A recombinant DNA molecule is made by joining together (a) a piece of foreign DNA (e.g. human DNA (b) vector DNA (e.g. a plasmid) The two DNAs can be joined together by an enzyme called DNA ligase

4. Cloning the recombinant DNA molecules

The recombinant DNA molecules will replicate if they are taken up by a bacterial cell and the bacterial cell will be transformed to antibiotic resistance.Cells that have not taken up a recombinant DNA molecule will be killed by the antibiotic.

Isolation of DNA from mRNA

DNA can be obtained from mRNA by hybridization method or by reverse transcritptase method.

Hybridization method
This method depends upon the principle that mRNA forms a complex with complementary segments of DNA from which they are transcribed. The total DNA from an organism is first isolated. This double stranded DNA treated with the heat or alkali and is converted into single stranded DNA by denaturation. This single stranded DNA is than mixed with mRNA transcribed by genes. The mRNA pairs with the complementary region of DNA to form DNA- RNA complex (hybrid). The complex is isolated and the DNA is separated and the DNA separated from RNA. The ssDNA which get separated has same base sequences as that of one base sequence from which mRNA was transcribed. This ssDNA can now be converted to dsDNA by polymerization action of DNA polymerase. This method is useful for isolating genes which exist in multiple copies e.g. ribosomal genes.

Cloning has 5 steps

1. Fragmentation 2. Ligation (Gluing together) 3. Transfection (Insertion into the cell) 4. Cloning / Multiplication 5. Screening / Selection

Fragmentation
1. DNA is isolated from the organism of choice (virus, bacteria, plants or animals )is cut into gene size pieces with restriction enzyme (endonucleases). The restriction enzyme cuts the DNA at specific points (Cohesive ends). These fragments are of different length. 2. On the other hand outer membrane of E. coli bacteria (host cell) is first dissolved by detergents like liquid and its DNA is released. The plasmid DNA is separated from

chromosomal DNA in an ultracentrifuge. Now plasmid DNA is placed in a solution containing restriction enzyme.

Ligation (Gluing together)

1. Foreign DNA segments are mixed with linear DNA. Random association between two types of DNA takes place by hydrogen bonding at cohesive ends. The foreign DNA inserted into plasmid DNA to form a linear ring or an a longer linear form.

2. Covalent joining is brought about by another enzyme called DNA ligase. 3. The resulting DNA is called plasmid chimera (Recombinant DNA). Both foreign and plasmid DNA joined to different combinations of fragments.

Transfection
The chimera plasmids are placed in a solution containing CaCl2 and normal E. Coli bacteria (Hersey et.al., 1952). On heating suddenly (2-5 min, 420C) or electroporation the E. coli membrane become permeable to plasmid chimera. Which pass into the cells thus recombinant DNA plasmid are taken by E. coli. Transfection can be done through--1. Lipid Mediated Method (Nelson et.al.,1993) 2. Calcium Phosphate Method (Graham and van der Eb, 1973, Loyter et al. 1982) 3. Electroporation (Wong and Neumann, 1982, Shigekawa and Dower, 1988)) 4. Polybrene (Polycations) (Nead et.al., 1995) 5. DEAE- Dextrom mediated (Di Ethyl Amino Ethylo Ethyl Dextron, Warden 1968, (Vaheri and Pagano, 1965; McCutchan and Pagano, 1968) 6. Virus (Vorburger and Hunt, 2002) 7. Biolistic (Gene gun, Microprojectile Bombardment, Klein 1987, Kikkart 2005) 8. LASER Transfection 9. Transfection Enhanced by Elevated temperature 10. Plant gene transfer (Campbell 1999, Lorence 2004) 11. Fine needle (Cappechi, 1980, Wu et al. 1998).

Cloning / Multiplication
The bacterial cells are spread on a nutrient medium in a petridish. Each E. coli cells containing particular gene inserted in its plasmid grows by repeated binary fission into colony. Every cell in a colony are genetically identical to each other and these individual are called clones. In favorable conditions each cell may produce 105 or 106 transformed clones.

Screening / Selection
Selection out the cells that are successfully transfected with new DNA.The bacterium E. coli divides every after 22 minutes under favorable conditions, theoretically giving rise to billions of cells in less than 11 hours. This will produce a large clones of recombinant DNA.

Genetic modification using recombinant DNA techniques allows us to move single genes whose functions we know from one organism to any other. In selective breeding, large sets of genes of unknown function are transferred between related organisms. By 2000 the human genome had been sequenced and today we use recombinant DNA techniques, in conjunction with molecular cloning to: produce new medicines and safer vaccines. treat some genetic diseases. enhance biocontrol agents in agriculture. increase agricultural yields and decrease production costs. decrease allergy-producing characteristics of some foods. improve foods nutritional value. develop biodegradable plastics. decrease water and air pollution. slow food spoilage. control viral diseases. inhibit inflammation.

Cloning Animals
List of all the animals humans have successfully created. This list has many great achievements that we have created over the years till 2009 Tadpole: (1952) Carp: (1963) Mice: (1986) Sheep: (1996) Rhesus Monkey:(January 2000) Gaur: (2001) Cattle: (males, 2001) and (2005)

Cat:(female, late 2001) Dog: (2005) Rat:(2003) Mule:(4 May 2003) Horse: (28 May 2003) Water Buffalo:(February 6, 2009) Camel: (2009)