Module 1.2 Basic Principles of DNA Recombinant Technology

• Formed by laboratory methods of genetic recombination and molecular cloning • Genetic material from multiple sources • Creates a DNA molecule that would otherwise not be found in nature

• Alteration of genetic material outside an organism and introduction of the altered DNA fragment into organisms via an appropriate vector • Goal is to obtain enhanced and desired characteristics in living organisms or as their product • Method of joining two or more DNA molecules o create a hybrid • the result is a DNA molecule that does not exist in nature

Restriction endonuclease and ligases

restriction endonucleases

Recognition sequence: sequence recognized by restriction endonuclease Restriction site: location along the DNA where the cut is actually made, may within or outside the recognition site

Types I

Type II (25 bp resolution); Type I (1, 000 bp resolution)

EcoR1 (G|AATTC) - from - E. coli R-strain HindIII (A|AGCTT) - from - Haemophilus influenzae D-strain 3

ligase

Deoxyribonucleases and Ribonucleases

Stanley N. Cohen

λdvgal 120 (isolated from bacteriophage lambda)

galactose metabolism

Simian Vacuolar Virus 40 (SV40) genome

plasmid-Stanley Cohen 101 (pSC101),

severe combined immunodeficiency disorder

Molecular cloning - creation of multiple copies of a fragment of DNA reproductive cloning - creation of an exact copy of the whole multicellular organism

recombinant DNA technology - a group of molecular techniques used to create recombinant DNA molecules by piecing together DNA fragments from multiple sources. The result is a chimeric DNA that is otherwise not found in nature Molecular cloning - is a molecular technique used to create multiple copies of DNA molecules with desirable sequences. Often, these DNA molecules are in the form of vectors containing a gene of interest. This vector is introduced in a cloning host, usually a bacterium or yeast, allowing production of the exact copy of the DNA. Genetic engineering is the process of using recombinant DNA technology to modify an organism’s DNA to achieve desirable traits. Addition of foreign DNA in the form of recombinant DNA vectors that are generated by molecular cloning is the most common method of genetic engineering.

• Isolate DNA containing gene of interest; isolate vector • Restriction digestion. Cut donor DNA and vector with the same restriction endonuclease. • PCR. After cutting, the donor DNA and the vector DNA can now be theoretically mixed to form a recombinant plasmid. However, some recombinant technology workstreams incorporate PCR to amplify the gene of interest after being subjected to restriction digestion such that a high number of the DNA of interest is produced. This ultimately increases the efficiency of the ligation process. • Ligation. In this step, the vector and the donor DNA are mixed to create a vector-donor hybrid by base-pairing of their sticky ends. The DNA hybrid is then covalently linked by DNA ligase that seals the nick in the sugar-phosphate backbone. • Insertion into cloning host. In this step, the recombinant plasmid is introduced into a cloning host (often bacterium such as E coli.) in a process known as transformation. The competency of the cloning host is one important factor to be optimized in this process, as incompetent cloning are less likely to take in the recombinant plasmid. To aid in the transformation process, techniques such as electrophoration, heat shock, or addition of Calcium phosphate are usually employed to increase the efficiency of transformation. • Isolation of transform cell. The result of transformation process is a mixture of transformed and untransformed cell. To isolate only the transformed cell, the cells are plated on a medium added with a particular antibiotic. The recombinant plasmid carried with it a selectable marker in the form a resistance genes against the antibiotic. As a result, only transformed cells that are expressing the recombinant plasmid can survive in the medium. • Expansion of the transformed cells. In this step, surviving colonies of transformed cells are expanded in large batch cultures. In the process, the recombinant plasmid is cloned in large amounts as the cells divides. • Isolation and cleaning of recombinant plasmid from expanded culture. • Transfer of recombinant plasmid in desired expression host. In this step, the extracted and cleaned recombinant plasmid is transferred to desired hosts to be expressed. The biological properties of the inserted gene is then tested. This may involve observing the phenotype of the cell or by extracting the expressed protein and subjecting it to some kind of functional assay.

polymerase

restriction enzymes

endonuclease - within sequence exonuclease - from ends

origin of replication selectable marker cloning sites

plasmid bacteriophages