RECOMBINANT DNA TECHNOLOGY

GENETIC ENGINEERING

• TECHNOLOGY THAT MANIPULATES OR MODIFIES THE GENE OF AN ORGANISM, WITH THE INTENTION OF CREATING IMPROVED PRODUCTS OR METHODS.

• TRANSGENIC PLANT/ANIMAL

• MAINLY USES TECHNIQUES OF DNA RECOMBINANT.

• A PROCESS THAT OCCURS RANDOMLY AND SPONTANEOUSLY IN NATURE.

• BUT NOW HAS BEEN MANIPULATED BY GENETICISTS.

DNA RECOMBINANT OCCURS NATURALLY THROUGH:

a) Crossing over during meiosis Ib) Sexual recombination when chromosomes

from two separate individuals combine to produce offspring.

c) Spontaneous mutationsd) Bacterial transformationse) Viral infections- Random and undirected (no specific goal)

TOOLS USED IN GENETIC ENGINEERING

1. RE

2. VECTORS

3. DNA LIGASE

4. HOST O/M

RESTRICTION ENDONUCLEASES/ ENZYMES

• Recombinant DNA technology developed from the discovery of certain bacterial strains which seemed immune to attack by bacteriophages.

• These strains produced special enzymes that restricted the replication of foreign DNA which had infected the bacterial cells. (hence they were called restriction enzyme)

• The site at which a restriction enzyme acts is called the restriction site. This site is typically 4-6 nucleotides long.

Types of recognition sequences

Blunt ended DNA

• Produced staggered cuts, sticky ends.

• Restriction site which produces sticky ended DNA after cleavage - palindromic

Sticky ended DNA

• This sticky ends are single stranded and they can pair with the complementary single stranded ends of other DNA molecules which have been cut with the same enzyme.

• Thus possible to bind DNA fragments from different sources.

This is called recombinant DNA

EXAMPLES OF RESTRICTION ENZYMES AND RESTRICTION SITES

A SINGLE RESTRICTION ENZYME MAY CUT A DNA MOLECULE IN SEVERAL PLACES BECAUSE THE TARGET SEQUENCE USUALLY OCCURS MANY TIMES IN A LONG DNA MOLECULE.

VECTORS• DNA molecules, derived from a plasmid or

bacteriophage into which DNA fragments may be inserted.

• A. Definition - Bacteriophage (phage) are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery (i.e., viruses that infect bacteria.).

• A 'plasmid' is a DNA molecule separate from the chromosomal DNA and capable of autonomous replication. In many cases, It is typically circular and double-stranded. It usually occurs in bacteria, and is sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae).

• This vector can then carry the foreign DNA into a host cell.

• Vectors unable to replicate independently (outside the cell)

Properties of Plasmids

1. Small molecules with known structures

2. Contain an origin of replication ---the plasmid can replicate itself in the host cell

3. Have one or more restriction sites recognised by RE

4. They code for special functions which confer well-defined features on the host, thus making them easier to be selected.

Examples of some vectors used in genetic engineering

• pBR322• pUC18• YAK• lambda phage

VECTORS

Cloning Vector

pBR322…..

Expression vectors…

Expression Vectors

Bacteria Artificial Chromosome (BACs)

Yeast Artificial Chromosomes (YACs)

Ti vectors- Naturally occuring plasmids (around 200 kb

in size- -isolated from bacterium Agrobacterium

tumefaciens, which is a soil borne plant pathogen that causes disease namely as crown gall disease.

DNA LIGASE• Required to anneal or join the sugar-

phosphate backbones in the recombinant DNA molecule.

HOST O/M• Vectors unable to replicate independently

(outside the cell)

• So they have to be taken up by a host o/m.

• E. coli is the most widely used host organism :

1) as its biological and genetic characteristics are well known

2) Produces rapidly

• Other host o/m – yeast and mammalian cells.

CLONING

• Cloning in recombinant technology is defined as the production of a cell line or culture, all of whose members contain identical copies of particular nucleotide sequence.

CLONING

FIVE BASICS STEP IN CLONING PROCESS:

ISOLATION OF TARGET DNA AND VECTOR DNA

Insertion of target DNA into vector DNA using RE and DNA ligase

Transformation/transduction of host cells.- Uptake of recombinant vector DNA by the host cells

Cloning of host cells carrying foreign genes- amplification.

Screening of cell clones carrying the gene interest

Example :

• The process of insulin production by E. coli as an example.

• Note that – the chances of success are very small !!!

STEP 1 (ISOLATION)

• Two kinds of DNA are prepared; 1. the gene of interest (insulin gene) which

is cultured from human cells, 2. bacterial plasmid with two particular

genes, that is the ampR gene and the lac Z gene.

• The ampR gene confers resistance to the antibiotic ampicillin. Thus bacteria with this plasmid can grow in media which contain ampicillin.

The lac Z gene codes for the synthesis of B-galactosidase which hydrolyses lactose.

The plasmid has a single restriction site recognised by the restricion endonuclease, and the site is situated in the lac Z gene.

STEP 2 (Recombination)• Half of the RE solution is mixed with the

human DNA• This will produce several hundred

thousand DNA fragments with sticky ends.• The rest of RE enzyme is then mixed with

the plasmid molecules and this will produce an equally large number of linear plasmids molecules with sticky ends complementary to those of the target DNA fragments.

• The human DNA fragments are mixed with the plasmid fragments ATP and DNA ligase are added.

• Since all the fragments have complementary sticky ends, at least three possible combinations may result from the pairing of these ends.

STEP 3 (TRANSFORMATION)

• The mixture of DNA molecules produced in step 2 is added to a culture of bacterial cells, usually E coli.

• Calcium ions are added ---stimulated to take up small pieces of DNA

• Only about 1% -----recombinant DNA.

STEP 4 (CLONING)

• The bacteria are plated onto nutrient plates which contain ampicilin and sugar called X-gal.

• Untransformed strains of E. coli will be killed by ampicilin. Only those cells which have acquired the plasmid with the ampR gene will be able to grow in the medium.

• P? Q? R?

• X-gal in the medium is used to indentify those colonies of bacteria with recombinant plasmids.

• X-gal is hydrolysed by B-galactosidase, an enzyme which is coded for the lac Z gene. Only bacteria with intact lac Z genes can produce this enzyme.

• B-Galactosidesidase hydrolyses X-gal and produce a blue compound, so bacteria colonies with functional lac Z gene will be blue.

• Bacteria colonies with recombinant plasmids will form white colonies.

• Having identified the bacteria colonies with the recombinant plasmids, the final step is to determine which of these colonies contain the DNA sequence which encodes insulin.

STEP 5 (SCREENING)• A nitrocellulose membrane or filter paper is

laid on the surface of a nutrient agar plate on which the bacteria colonies with recombinant plasdmids have been cultured.

• Bacteria cell will be transferred to the filter.• The filter is treated with sodium hydroxide

solution which denatures the DNA by breaking the hydrogen bonds between the two polynucleotide strands.

• This leaves single-stranded DNA on the filter. Single-stranded DNA will pair up (hybridise) with any nucleic acid molecule with a complementary sequene of bases.

• The filter is incubate with a solution containing gene probe molecules. The gene probe for insulin is easily obtained by synthesising a short sequene of nucleotides which corresponds to the sequence of animo acids in insulin.

• The radioisotope 32p is used in this process so that the gene probe is radioactive.

• Wherever the gene probe meets a complementary DNA sequence on the nitrocellulose filter, it will bonds with it, forming a DNA-probe hybrid.

• The location of these hybrid is determined using autoradiography and by comparing the autoradiogram with the original master plate, those bacteria colonies with the insulin gene are selected.