GENETIC ENGINEERING (RECOMBINANT DNA TECHNOLOGY)

GENETIC ENGINEERING: technology that manipulates/ modifies the gene of an organism, with the intention of creating improved products or methods, such as transgenic plant/animal. mainly uses techniques of DNA recombinant. DNA recombinant is a process that occurs randomly and spontaneously in nature. but now it has been manipulated by geneticists

DNA RECOMBINANT OCCURS NATURALLY THROUGH: Crossing over during meiosis I Sexual recombination when chromosomes from two separate individuals combine to produce offspring. Spontaneous mutations Bacterial transformations Viral infections - Random and undirected (no specific goal)

Gene Cloning: isolation and making of many copies of a gene 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.

TOOLS USED IN GENETIC ENGINEERING RE (restriction endonuclease/ enzyme) vectors DNA ligase host organism

Vector A small piece of DNA where a gene of interest is introduced (vector acts as a gene carrier). The vector is then placed within a living cell (host cell), where it replicates and produces multiple identical copies of the inserted gene. One of the most commonly used vectors are plasmids.

Plasmids as vector Plasmids are small circular pieces of DNA that are found in bacterial and some eukaryotic cells. Plasmids contain origins of replication that are recognized by the host cell. Many plasmids contain selectable markers, can grow in the presence of an antibiotic or other toxic substance.

Vectors unable to replicate independently (outside the cell)

Properties of Plasmids Small molecules with known structures Contain an origin of replication ---the plasmid can replicate itself in the host cell Have one or more restriction sites recognised by RE They code for special functions which confer well-defined features on the host, thus making them easier to be selected.

RESTRICTION ENDONUCLEASES/ ENZYMES (RE) REs recognise base sequences and then cleave the DNA at two defined locations . The site at which a restriction enzyme acts is called the restriction site. This site is typically 4-6 nucleotides long. RE is found in many bacterial species, where they protect the organism from the invasion of foreign DNA REs usually recognise palindromic sequences.

Some REs produce fragments with “sticky ends,” where they will form hydrogen bond with each other due to their complementary sequences. 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 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.

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

EXAMPLES OF RESTRICTION ENZYMES AND RESTRICTION SITES

DNA LIGASE Required to anneal or join the sugar- phosphate backbones in the recombinant DNA molecule to attach the target DNA (gene of interest) with vector, becoming recombinant DNA.

HOST ORGANISM Vectors (such as plasmids) are unable to replicate independently (outside the cell) So they have to be taken up by a host organism. E. coli is the most widely used host organism: as its biological and genetic characteristics are well known Produces rapidly Other host o/m – yeast and mammalian cells.

FIVE BASICS STEP IN CLONING PROCESS: THE 5 STEPS OF GENETCI EGNINEERING USING RECOMBINAT DNA 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.

STEP 1 (ISOLATION) Two kinds of DNA are prepared; the gene of interest (insulin gene) which is cultured from human cells, bacterial plasmid with two particular genes, that is the ampR gene and the lac Z gene. The ampR gene give resistance to the antibiotic ampicillin. 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 restriction endonuclease, and the site is situated in the lac Z gene.

STEP 2 (Recombination) Half of the RE solution is mixed with target DNA fragment which produce insulin (target DNA). This will produce several hundred thousand DNA fragments with sticky ends. The rest of RE enzyme is then mixed with the plasmid molecules  large number of linear plasmid molecules with sticky ends complementary to the target DNA fragments will be produced.

The target DNA fragments are mixed with the plasmid fragments, then 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 (host), usually E. coli. Calcium ions are added to stimulate uptake of plasmids by host cells.

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.

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.

STEP 5 (SCREENING) 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. nitrocellulose membrane or filter paper is laid on the surface of a nutrient agar plate on which the bacteria colonies with recombinant plasmids have been cultured. Bacteria cell will be transferred to the filter.

STEP 5 (SCREENING) The filter is treated with sodium hydroxide (NaOH)  denatures DNA by breaking the hydrogen bonds between t 2 polynucleotide strands. Single-stranded DNA will pair up (hybridise) with any nucleic acid molecule with a complementary sequence of bases. The filter is incubated with a solution containing gene probe molecules. *gene probe for insulin is easily obtained by synthesizing a short sequence of nucleotides which corresponds to the sequence of amino acids in insulin.

The radioisotope 32p is used in this process so that the gene probe is radioactive. gene probe will bind to complementary DNA sequences on the filter, 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.