Lab Techniques and Biotechnology

Lab Techniques and Biotechnology

1. gel electrophoresis: separating nucleic acids or proteins based on molecular weight/size and charge. Electric current is applied to gel, fragments are loaded into gel. DNA is negatively charged and moves towards the anode (positively charged end). Smaller fragments move lower and larger fragments remain stuck at the top.

   • isoelectric point: the pH at which the protein has no net electric charge; influenced by the ionic character (partial charge?) of the side chains in the protein at a certain pH. This property of proteins can be used as a sepeartion technique, where proteins are moved over a pH gradient gel, and the proteins at their isoelectric point may precipitate out due to lowered solubility.
   • Southern blot: when DNA fragments are transferred to a nylon membrane and are probed with a fluorescent probe for certain sequences

2. PCR (polymerase chain reaction)

   • DNA, primers, and taq polymerase are mixed together. 3 step process- 1} DNA is denatured into single strands by heating. 2} temperature is cooled so primers bind to their complementary strands. 3} taq polymerase makes new strands which are amplified into millions of strands as the steps are repeated over the multiple cycles.
   • qPCR (quantitative, aka real-time PCR), quantifies the reaction in real time by watching the reaction on camera and seeing the fluorescence increase
   • Short tandem repeat (STR) analysis involves analyzing smaller samples of DNA that are amplified through PCR to compare the number of base pair repeats at specific loci.

3. DNA sequencing

   • southern blotting: restriction enzymes cut DNA into fragments. The fragments are seperated on a gel. The seperated fragments are denatured (become single stranded). The denatured fragments, still in the gel, are put into a membrane. Then a hybridization probe (a radioactive or fluoroscently labeled fragment of DNA or RNA that anneals or hybridizes to a complementary fragment to form a ds helix) is applied to find a certain sequence.
   • shotgun sequencing and high throughput sequencing: use restriction enzymes to fragment DNA prior to sequencing, and decrease time and money involved
   • Sanger Sequencing: individual pieces of DNA are sequenced through PCR. DNA nucleotides and dideoxynucleotides + primer + DNA polymerase + template DNA are required. PCR reaction is started and polymerase stops adding nucleotides after adding dideoxynucleotides (they do not have OH group at the 3' C, which is required to form phosphodiesterase bond). The fragments are labelled with dyes based on their final nucleotides. The fragments are run through capillary gel electrophoresis to separate them, and laser is applied to detect color of dye as the fragments pass. The sequence is then decoded using the fluorescence patterns. Used on DNA up to 900 base pairs long, and often used for bacterial plasmid sequencing.
     • aka chain termination reaction (now considered outdated d/t cost and less efficiency)
   • next-gen sequencing: modern methods developed after Sanger sequencing; they feature highly parallel (many reactions occurring at the same time), low-cost, and fast methods. They are usually applicable on DNA 50-700 nucleotides long

4. Northern blots are used to run mRNA samples on gels, DNA microarrys give the expression levels of certain genes, and rtPCR is used to detect RNA expression levels. Any of these methods could provide the relative abundance of a particular mRNA.

5. recombinant DNA

   • a rapidly expanding technology that can be used for: pharmaceutical development, gene therapy, medical applications, forensic analysis, agricultural applications, bioremediation, etc.
     • However, there are ethical concerns: consequences of the potential release of rDNA into nature, interaction of created recombinant organisms with wild type organisms, safety of recombinant organisms in the food chain, and the ethics of altering the DNA of organisms.
     • DNA technology has been used widely in agriculture. Some plants express a transgenic gene to prevent pests, which reduces the need of pesticides. Some plants express genes decreasing their risk of developing or catching certain diseases, which increases crop yield. If plants had the ability to fix their own nitrogen, this could save farmers money on fertilizer.
   • DNA molecule comntaining DNA from 2 or more sources
     • an organism with rDNA is called transgenic
       • Genetically modified organisms have been used to clean up the environment. Genetically modified bacteria are used to extract heavy metals from the environment, which is useful in the mining industry as well. Genetically modified pigs have an enzyme in their saliva that breaks down indigestible phosphorus, which helps clean water.
   • detected through gel electrophoresis and southern blotting
   • recombinant DNA is useful in drug production (recombinant plasmid is created and transformed into bacteria, the gene is expressed, the protein or molecule is then harvested) and vaccine production (surface protein of a harmful pathogen can be cloned into a harmless virus which is used to stimulate the human immune system via inoculation).

6. Gene cloning: a scientific technique where multiple copies of the gene are generated. This is often needed to isolate a gene from other DNA regions that may interact with it in order to study the function of said gene.

   • This requires a cloning vector. The most common example includes bacterial plasmids (small, self-replicating loops of DNA found naturally in bacterial cells—can be vectors carrying foreign genes).
     • viruses can also be vectors to introduce foreign genes into the genome of an organism
       • when choosing a vector or host organism, it is improtant to consider the desired protein's functions and the organisms capacity. For example, bacteria do not have post-transcriptional modification machinery, which is essential for the expression of many eukaryotic genes. Therefore, for such genes, it is better to use yeast as the host, as they are also microorganisms that can mass produce cheaply
     • This is due to a special feature that plasmids have called the multiple cloning site. This site, a DNA sequence with multiple sites, allows DNA fragments to be introduced into the plasmid via cutting by restriction endonucleases.
       • As a result, such plasmids are known as recombinant DNA because they are not found in nature in this form. The fragment of DNA that is introduced into the plasmid is known as a transgene. The goal is for the plasmid and the host DNA and the transgene to proliferate as the bacterial cell reproduces.
         • The proteins expressed from transgenes or recombinant DNA are known as recombinant proteins. However, not all plasmids are capable of expressing genes and the vector may need to be moved into a more conducive host. As with naturally occurring genes, environmental factors may influence the expression of the transgene; this component also allows researchers to study the expression of certain genes by manipulating the environment.
     • brought into bacterial cells from their environment naturally by process of transformation

7. restriction endonuclease: found naturally in bacteria, where they function as a defense mechanism to disable foreign DNA; each has a specific recognition sequence at the which they cut at the restriction site (they read and cut at a specific DNA sequence).

   • sticky ends: cohesive ends, stretch of unpaired nucleotides in the end of a DNA molecule, allowing pairing with another molecule; conducive for recombinant DNA formation.
   • blunt ends: non-cohesive ends, both strands terminate in a base pair, does not allow pairing with another molecule
   • RFLPs (restriction fragment length polymorphisms) are the difference in individuals of how restriction enzymes cut their DNA. These, along with variable number tandem repeats, can be analyzed in the DNA left behind at crime scenes to identify suspects or perpetrators.

8. DNA libraries

   • collection of DNA fragments stored and propagated in a population of micro-organisms through molecular cloning (assembly of recombinant DNA molecules perpetually replicated within host organisms)
   • genomic library: collection of the total genomic DNA from a single organism. The DNA is stored in a population of identical vectors, each containing a different cut/fragment of DNA.
   • complementary DNA library: DNA complementary to the mRNA taken from a particular organism, that is stored in vectors. Allows researchers to study genes that were actively being expressed under specific conditions
     • 1st reverse transcriptase makes a hydrid RNA + DNA molecule by synthesizing a complementary DNA strand to the mRNA. It has RNAse actiivty to degrade the RNA, and DNA polymerase activity to synthesize the 2nd DNA strand. Finally results in double stranded cDNA
     • prior to modern methods, insulin was produced in high quanities in bacteria using cDNA libraries
   • mutant libraries: used to determine the function of genes. Formed by forcing mutations in the population of the library. The library is then screened and differences in function are compared.
     • Knockout mutants: altered to result in loss of function of a gene. Knockout is then compared to wild type to see differences in phenotype and functioning, and to determine gene function

9. Methods of detecting gene expression

   • Western blots: use protein-specific antibodies to measure the relative abundance of proteins. Gel electrophoresis is used to separate the proteins, then they are transferred onto a solid matrix. Proteins on the matrix are exposed to antibodies, and they are visualized via fluorescence or enzymatic reaction.
   • in situ hybridization: study gene expression in a tissue or embryo. Tissue is plated and viewed under microscope. Membrane is opened through permeabilization. Probe is applied, and it binds to transcript of interest. Antibody is added and it binds to the probe. Then the substrate of the enzyme-antibody is added, and the antibody-probe-transcript complex is detected by the substrate. This method is used to id where transcripts are expressed.
   • Immunohistochemistry: antibody is used to target a certain gene and measure its expression. Then, a 2nd, labelled (with fluorescent molecule or enzyme) antibody binds to the first antibody and this measures expression. Ex: used in detecting certain diseases like cancer (measuring HER2 expression in a breast biopsy, colon cancer screening through FIT)
   • flow cytometry: cell samples are stained for protein markers using specific, fluorescently marked antibodies. The cells pass through a laser and they emit light as they pass. The light is measured, to analyze cell size and how many cells express the specific proteins being targeted. Many different proteins can be analyzed within one sample because the cytometers have multiple channels.

10. Methods of determining gene function:

    • Discovered genes can be compared to the genes of other organisms that they have similar sequences to.
    • Gene function can be determined by observing protein pathways. Immunoprecipitations are used to find protein binding partners. Cell lysates are exposed to specific antibodies to form complexes, then an antibody binding protein is added. The solution is centrifuged to precipitate out the complex bound to the antibody binding protein. After purification, the isolated proteins are analyzed through western blot or mass spectrometry. The data is put into gene network maps which demonstrate how the proteins from the target genes function together within a cell.
    • Location can be used to determine protein/gene function. The target gene is tagged to determine where it is expressed. Example: a target gene could be expressed in a cloning vector and linked to a fluorescent molecule like GFP, and wherever fluorescence is seen under a fluorescent microscope would be where the gene is expressed in the cell which could help determine its function.

11. Ethical concerns

    • Gene therapy has ethical concerns regarding unknown complications of introducing foreign genes into the human body.
      • Gene therapy uses a virus vector to deliver modified genes to target a defective or missing gene. If successful, it can be used to treat sickle cell anemia or Parkinson's disease.
    • Genetically modified foods and plants could potentially be harmful to both humans and the ecosystem at large.
    • Germ line therapy is controversial due to the implication of eugenics (purposefully modifying the human genetic makeup for certain attributes).
    • Stem cell harvesting is controversial due to the need to harvest cells from a blastocyst, which destroys the fetus.

12. microarray (DNA chip): monitor changes in gene expression based on cellular environment. The chip is spotted with samples of genes, then exposed to a cDNA sample. Any spot where the cDNA hybridizes on the chip indicates expression of that gene. Microarray technology is used to compare gene expression under varying conditions, has been essential in discovery of which genes are expressed or not in cancer, and can also be used to determine gene function.

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