Isolating Genes By Allison Michas and Haylee Kolding

Why isolate genes? Isolating genes allows scientists to test which genes cause certain effects. For example, scientists have been able to identify genes that increase risks for cancer, make artificial proteins for vaccines, and make discoveries about the process of eukaryotic gene expression. Isolating genes is necessary for the genetic engineering of certain traits.

History 1900s - First isolation of DNA when biochemists isolated DNA and RNA from cell nuclei. When observing the polymeric nucleic acids, they realized that there were two different types of nucleotides. One contained ribose and the other deoxyribose. This discovery led to the identification of DNA and a substance distinct from RNA Stuart Linn and Werner Arber succeed in isolating an enzyme that could selectively cut DNA

History Frederick Sanger was the first to fully sequence a DNA genome and create a DNA library Kary Mullis invented the PCR technique while working as a chemist at the Cetus Corporation in California Cetus Corporation joined with Perkin-Elmer Corporation to develop the DNA Thermal Cycler to regulate temperatures of the enzymes involved in PCR

DNA Libraries - Procedure DNA libraries are a collection of the total genomic DNA from a single organism. The DNA fragments from the organism are stored in bacterial plasmids in this procedure. 1. Extract and purify DNA 2. Digest DNA with restriction enzymes which cut the DNA at specific sequences 3. Insert DNA into plasmids -plasmid: type of vector that allows the DNA to be shuttled into bacteria 1. Plasmids are digested with restriction enzymes and then sealed to human DNA using DNA ligase -these molecules are recombinant 1. Make bacteria take up recombinant DNA 2. Bacteria reproduce and form more recombinant DNA This process allows researchers to analyze human DNA fragments and locate fragments that contain the genes they want to study.

Polymerase Chain Reaction (PCR) - Procedure PCR is typically used to make many copies a specific gene so that the function of that gene can be studied. DNA primers are used to mark the region of DNA to be amplified. PCR follows 3 steps: °C Denaturation - heat the DNA to a high temperature to separate the two strands of DNA 2. Annealing (at ~ °C) - reduce the temperature so that added DNA primers can bind to either end of the gene that you want to isolate 3. Extension - raise the temperature to about 70 °C to activate DNA polymerase to replicate the strands 4. Repeat - Each step doubles the amount of DNA copies of the target gene repetitions can produce billions of copies

PCR - Procedure PCR temperatures are regulated by a thermocycler to help the PCR process to continue. At the end of the process, billions of copies are made of a specific gene PCR also allows DNA to be sequenced

Current Research and Advancements DNA Libraries Today, DNA libraries are built from BACs, or bacterial artificial chromosomes, based on the F-plasmid of E. coli. BACs are artificially constructed and are often used to sequence the genomes of organisms. Since BACs are stable, easier to handle, and are able to accept large DNA inserts, they are now the preferred vector for building DNA libraries of complex organisms. Polymerase Chain Reaction (PCR) A modified version of PCR is being developed as an improved method for isolating microsatellites, or repeating DNA segments. This type of PCR is called Vectorette PCR, and involves the use of different primers. Vectorette PCR is useful when isolating homologous genes of related species.

Future Possibilities Right now, with gene isolation and genetic engineering technology, scientists have the ability to create “designer babies”. Gene isolation lets scientists pick and choose which specific genes to add or remove from a DNA sequence. This technology could be used to prevent genetic diseases from being passed on, but it is still highly controversial to genetically engineer humans. Meanwhile, this genetic engineering technology has been used to make stronger crops, mosquitoes that cannot spread malaria, crops that are used to fight malnutrition & produce vaccines, and much more. In the future, gene isolation will be used like it is used now: to make changes in a DNA sequence to change the way an organism works.

Sources Cloning%20a%20Gene