Recombinant DNA Technologies

Flashcards on Recombinant DNA Technologies

Genetic Engineering

Systematic operations on the DNA of organisms to perform genetic analysis or develop organisms with desired characteristics.

Alternative Names for Genetic Engineering

Recombinant DNA technology, gene cloning, DNA cloning, genetic manipulation/modification

Recombinant DNA Technology

Techniques to introduce a gene isolated from an organism into a host to reproduce and sometimes express it.

Recombination

The genetic basis of recombinant DNA technology.

Recombinant DNA

A combination of DNA molecules that do not naturally occur together.

Transgenic organism

A living organism altered by transferring genes from another species.

Why is gene cloning needed?

To obtain large numbers of copies of gene-sized DNA fragments.

Most widely used prokaryotic host

E. coli

EcoRI (restriction enzyme)

Cuts the DNA molecule into a linear shape with sticky ends.

Restriction enzymes

Break the sugar-phosphate backbone of DNA.

DNA ligase

Joins DNA fragments at their blunt or sticky ends.

The Stages of Cloning

DNA isolation, Integration of the target DNA into vector, Transfer of recombinant DNA into the host cell, Selection of host cells including recombinant DNA, Purification of the desired protein from host cells

Required Components for Gene Cloning

Target DNA, Restriction endonucleases, Vector, Ligase enzyme

Enzymes Used In Gene Engineering (4 main classes)

Nucleases, Ligases, Polymerases, Modifying enzymes

Types of Cloning

Cloning at the DNA / Gene level, Cloning at the cell level, Cloning at the organism/nucleus level

For Successful Gene Cloning (gene)

Must be able to replicate independently, transferable to the host cell, allow for selection

Plasmids

Double-stranded DNA molecules found naturally inside bacterial cells, replicating autonomously, outside the bacterial chromosome.

Types of Plasmids

Fertility (F plasmids), Resistance (R plasmids), Colicin (Col plasmids), Degradative plasmids, Virulence plasmids

Criteria for successful plasmid development

Small size (smaller than 15 kb)

Types of Viral Vectors

Retroviral vectors, Adenoviral vectors, Adeno-associated virus, Polio virus, Sindbis virus

Retroviruses

Synthesize their ds DNA using the enzyme reverse transcriptase and thus, integrate into the host genome.

Viral vectors

Can easily enter the cell and integrate their genetic material into the target cell.

Disadvantages of Viral Vectors

Inability to infect non-dividing cells (retrovirus), Adverse immunological effects (adenovirus), Cytotoxic effects (herpesvirus), Limited capacity to carry foreign genetic material (adeno- associated virus)

Methods of Integration of Recombinant DNA Molecules into Host Cells

'Transformation' if the carrier DNA is a plasmid, 'Transfection’ if the carrier DNA is a virus, Microinjection, Biolistics, Electroporation

Recombinant DNA Technology Methodologies

Fluorescent In situ Hybridization (FISH), Polymerase Chain Reaction (PCR), Reverse transcription, CRISPR / Cas9 System, DNA Sequencing

Areas Where Recombinant DNA Technology Is Used

Prenatal diagnosis, Cancer diagnosis, Vaccine production