Applied Biology Flashcards
GE: Add a foreign gene
A novel (foreign) gene is inserted from another species. This will enable the GMO to express the trait coded by the new gene. Organisms genetically altered in this way are referred to as transgenic.
GE: Altering an existing gene
An existing gene may be altered to make it express at a higher level (e.g. growth hormone) or in a different way (in tissue that would not normally express it). This method is also used for gene therapy.
GE: Delete or “turn off” a gene
An existing gene may be deleted or deactivated to prevent the expression of a trait (e.g. the deactivation of the ripening gene in tomatoes)
Restriction enzymes
One of the essential tools of genetic engineering is a group of special restriction enzymes (also known as restriction endonucleases). These can cut DNA molecules at very precise sequences of 4 to 8 base pairs called recognition sited. These enzymes are “molecular scalpels” that allows genetic engineers to cut up DNA in a controlled way. Before being isolated in 1970, the enzymes were discovered earlier in many bacteria. By using a “tool kit” of over 400 restriction enzymes recognizing about 100 recognition sites, genetic engineers can isolate, sequence and manipulate individual genes derived from any type of organism.
Ligation
DNA fragments using restriction enzymes may be reassembled by a process called ligation. Pieces are joined together using an enzyme called DNA ligase. DNA of different origins produced in this way is called recombinant DNA (because it is DNA that has been recombined from different sources). The combined techniques of using restriction enzymes and ligation are the basic tools of genetic engineering / recombinant DNA technology.
Plasmids
- Plasmids are small, circular, self-replicating DNA molecules separate from the main bacterial chromosome
- They carry only a small number of genes, among them often antibiotic resistance genes that provide the bacterium with resistance against particular antibiotics
- They can move between cells, and even between species, by conjugation.
- They can be easily isolated from bacteria and manipulated to form recombinant plasmids
- They can be reintroduced into bacterial cells by adding them to a bacterial culture medium; under suitable conditions, some bacteria will take up the plasmid from the culture medium by the process of transformation (=the transfer of DNA to any living cell).
- Bacterial cells reproduce rapidly and, in the process, multiply any foreign DNA they carry.
The plasmids used for genetic engineering:
Are often made artificially
Carry two marker genes that provide bacteria either both with resistance to antibiotics like ampicillin (ampR gene) and tetracycline (-> tetR gene) or only with resistance to one antibiotic and the other marker gene encodes for a certain gene product (e.g. lacZ gene encodes for ß-galactosidase; ß-galacrosidase hydrolysis a mimic of lactose (X-gal) to form a blue product).
Have only one restriction (enzyme recognition) site for each restriction enzyme; the restriction sites always lie within one of the marker genes, e.g. within the tetracycline resistance gene -> this is important for identifying transformed bacteria!
Carry an origin of replication (ori) for independent reproduction
How to reproduce recombinant DNA
If two pieces of DNA are cut by the same restriction enzyme, they will produce fragments with matching sticky ends (ends with exposed nucleotide bases at each end).
When two such matching sticky ends come together, they can join by base pairing. This process is called annealing. This can allow DNA fragments from a different source, perhaps a plasmid, to be joined to the DNA fragment.
The joined fragments will usually form either a linear molecule or a circular one, as shown here for a plasmid. However other combinations of fragments can occur.
The fragments of DNA are joined together by the enzyme DNA ligase, producing a molecule of recombinant DNA.
Detection of recombinant pacteria
- A plasmid vector with 2 marker genes
- There is a restriction site within one of them
- cDNA of the desired human gene has the same restriction sites on both of its ends
- both is out with the same restriction enzyme
- foreign DNA is inserted, old gene is inactivated
- host bacteria are cultivated
Detection of recombinant bacteria:
- Transformation of a bacterium with a recombinant plasmid is very rare
- Duplicating pads test the colonies on various nutrient mediums to determine one of the following cases: Bacterium without plasmid; bacterium with non-recombinant plasmid; Bacterium with recombinant plasmid
e. g. the blue white screening in which the lac z gene in the plasmid makes blue colonies, but if its disrupted by the inserted Insulin gene, it’s white.
Other methods of gene tranfer: Transformation
In transformation, the genotype and phenotype of a prokaryotic cell are altered by the uptake of foreign DNA from its surroundings. For example, bacteria from a harmless strain of Streptococcus pneumonia can be transformed to pneumonia-causing cells if they are placed into a medium containing dead, broken-open cells of the pathogenic strain. This transformation occurs when a live non-pathogenic cell takes up a piece of DNA carrying the allele for pathogenicity. The foreign allele is then incorporated into the cell’s chromosome, replacing the existing non-pathogenic allele – an exchange of homologous DNA segments. The cell is now a recombinant: Its chromosome contains DNA derived from two different cells. In biotechnology, this can be used to introduce foreign genes into the E.coli genome – genes coding for valuable proteins, such as human insulin.
Other methods of gene tranfer: Transduction
In transduction, bacteriophages (or phages) carry bacterial genes from one host cell to another. For most phages, transduction results from accidents that occur during the phage reproductive cycle. A virus that carries bacterial DNA may not be able to reproduce because it lacks its genetic material. However, the virus may be able to attach to another bacterium (recipient) and inject the piece of bacterial DNA acquired from the first cell (the donor). Some of this DNA may subsequently recipient cell’s chromosome by DNA recombination. In such a case, the recipient cell’s chromosome by DNA recombination. In such a case, the recipient cell’s chromosome becomes a combination of DNA derived from two cells: genetic recombination has occurred.
Other methods of gene tranfer: Conjugation and Plasmids
In a process called conjugation, genetic material is transferred between two bacterial cells (of the same or different species) that are temporarily joined. The DNA transfer is one-way: One cell donates DNA, and the other receives it. The donor uses sex pili to attach to the recipient. After contacting a recipient cell, each sex pili retracts, pulling the two cells together. A temporary cytoplasmic “mating bridge” then forms between the two cells, providing an avenue for DNA transfer. In most cases, the ability to form sex pili and donate DNA during conjugation results from the presence of a particular piece of DMA called the F factor (F for fertility). The F factor consists of about 25 genes, most required for the production of sex pili. The F factor can exist either as a plasmid or as a segment of DNA within the bacterial chromosome.
Function and steps of PCR
The function of the polymerase chain reaction (=PCR) is to copy (amplify) any specific segment (-> target sequence) of a DNA sample completely in vitro. This might be used when there is little DNA available from the source (e.g. in a crime scene or from a long-extinct species), making it impossible for modern-day DNA technology to work with without upon the quantity of DNA available.
Step 1: Denaturation: Heat the reaction mixture to 95 degrees Celsius. The heat denatures the DNA, breaking the hydrogen bonds that hold the strands together.
Step 2: Annealing: Reduce the temperature to around 60 degrees Celsius so that the primers can form hydrogen bonds, or anneal, with their complementary sequences in the target DNA.
Step 3: Polymerization/Extension: Raise the temperature to 72 degrees Celsius. Taq polymerase functions optimally at this temperature and begins polymerization, adding nucleotides to the 3 prime ends of each primer attached to the DNA strand.
Repeated again and again (33 cycles yield over 109 copies)
Role of Primer
The starting point for the DNA polymerase to attach nucleotides to its 3 Prime ends.
With their length of 15 to 30 bases, they will only bind to a single of DNA in an organism’s genome.
Role of thermostable DNA polymerase
Thermostable to withstand the heat, DNA polymerase adds nucleotides to the three-prime end of each primer to elongate extend the two new target sequences for the process to begin again.
