Gene manipulation

Gene Manipulation

from Campbell's Biology, Benjamin/Cummings Publishing Co., 1990 and Wallace, King and Sanders' Biology: The Science of Life, Scott, Foresman and Co. 1986.

Basic Strategies of Gene Manipulation

Applications of Recombinant DNA technology

Safety and Policy Matters

Return to HW Page

Recombinant DNA technology is a set of techniques for recombinant genes from different sources and transferring the recombinant DNA into other cells, where it may be expressed.
Such gene manipulation has sparked an explosion of discoveries in molecular biology and an industrial revolution in biotechnology

Basic Strategies of Gene Manipulation

Before the advent in 1975 of recombinant DNA technology molecular genetics were largely limited to studying laboriously obtained mutant organisms. Only phage and prokaryotic genes were readily transferable from one cell to another, by use of the natural processes of transformation, conjugation, and transduction.
Recombinant DNA technology has now made it possible to study virtually any gene in detail by using one or more of a number of biochemical tools and methods.

A variety of bacterial restriction enzymes recognize short, specific nucleotide sequences in DNA and cut the sequences at specific points on both strands to yield a set of double stranded DNA fragments with single "stick-ends", which readily form base pairs with complementary single stranded segments on other DNA and purify these individual fragments.

Recombinant DNA DNA molecules can be introduced into host cells by means of bacteriophage or plasmid vectors. in either case, gene cloning results when the foreign genes replicate inside the the host bacterium.

The two major sources of desired genes for insertion into vectors are

(a) genomic libraries contain that all the plasmid or phage-carried DNA segments isolated directly from an organism.

(b) complementary DNA synthesized from mRNA templates. The latter source is especially useful for manipulating and expressing intron-rich eukaryotic DNA.

A useful tool in Genetic engineering is gel electrophoresis. This techinique separates particles based on size.

The presence of a particular gene can be determined directly, using radioactivity labeled nucleic acid segments of complementary sequence called probes. Detection can also be done indirectly by identifying the protein product of the gene.
Machines are now used to synthesize short genes of known nucleotide sequences long stretches of DNA, using restriction enzymes and gel electrophoresis as key tools. DNA sequences collected in computer banks allow rapid analysis and automatic translation into amino acid sequences.
Bacteria and yeast have been successfully used to make the gene products of most recombinant DNA. However, antibodies, requires the use of cultured animal and plant cells that posses the requisite complex biosynthetic machinery.

Applications of Recombinant DNA technology

Recombinant DNA technology has been a boon for biological research, allowing investigators to answer questions about molecular evolution, probe details of gene organization and control, produce and catalog proteins of interest, and map eukaryotic genes.
The human genome project involves linkage mapping, physical mapping, and sequencing of the human genome as well as similar analysis of the genome of some sample species.

Restriction fragment length polymorphisms are differences in DNA sequence on homologous chromosomes that result in different patterns of restriction fragment lengths, which are visualized as bands upon gel electrophoresis after treating the DNA fragments with restriction enzymes. RFLPs can be used as markers for genetic mapping and for fingerprinting DNA for medical or forensic purposes.

Polymerase chain reaction (PCR) is a technique for quickly amplifying DNA in vitro.

Exciting medical applications of recombinant DNA technology include the large-scale production of previously scarce pharmaceutical products; the design of safer, more effective vaccines; the development of diagnostic tests for detecting mutations that cause genetic disease; and the ultimate prospect of curing and preventing genetic disorders caused by single defective genes. The last application faces a host of formidable technological and ethical challenges that have yet to be resolved.
Potential agricultural benefits of genetic engineering include the design of more productive and disease-resistant plants and animals and improvement in food quality.
Plant cells and amenable to gene manipulation, using the Ti plasmid from Agrobacterium as the principle vector.
Preliminary results with engineering of single gene traits in plants have created optimism for increasing crop yields by biotechnology. Increasing biological nitrogen fixation by gene splicing promises to have tremendous potential for increasing the worlds food supply.

Safety and Policy Matters

When scientists realized that recombinant DNA technology might have some potentially dangerous consequences, they outlined a set of safety measures that have been implemented in laboratories across the country.
Several U.S. government agencies are involved in regulating the use of recombinant DNA technology.