Double Stranded Rna Synthesis Lab Report

The objective of these labs were to use sophisticated techniques to produce double-stranded RNA that was incubated with live Drosophila cells to inhibit the expression of our two genes of interest. The overall process of the four labs was to isolate and amplify DNA using the polymerase chain reaction with primers that contained gene-specific sequences to Thread or Dynamin-related protein 1 (drp-1), along with T7 promoter site sequences. The amplified DNA was purified and both strands of the DNA were used as templates for in vitro transcription reactions, producing complementary RNA strands for each of the two genes. The RNA was heated, to separate the RNA hybrids and then the complementary strands were allowed to anneal, forming double-stranded RNA molecules. The purified double-stranded RNA produced in lab was then used with THREAD or drp-1 to cause RNA interference in Drosophila cells. Finally, we examined these cells and photographed them using inverted microscopes.
In a natural environment replication is initiated when DnaA proteins bind DnaA boxes, separating the AT-rich region of the DNA.
This allows Helicase to enter and break hydrogen bonds in the 5’ to 3’ direction. Single stranded binding proteins hold the DNA in place while DNA polymerase adds RNA primer, and DNA polymerase III synthesizes 5’ to 3’. In the lab we manipulated this process by denaturing the hydrogen bonds in a solution heated up to 95 °C. We then annealed the primer by placing the DNA in a solution to 45- 65 °C. This allowed DNA primers to bind to the template strand. Finally, we heated the solution to 72 °C to allow DNA polymerase to replicate a new complimentary strand to the
template.
Transcription was then replicated in the lab in order to complete the experiment. Transcription is a process that requires the enzyme RNA polymerase. In addition, it requires a DNA template that has included in its sequence a specific stretch of nucleotides called a promoter site. This enzyme can pull apart DNA at the promoter and start transcription of DNA into RNA. When RNA polymerase binds at the promoter site and pulls apart the DNA, it uses the strand oriented in the 3’ to 5’ direction as a template for the process and begins the construction of a complementary sequence of RNA in the 5’ to 3’ direction until a termination sequence is reached and the RNA polymerase and newly synthesized RNA molecule are released. In lab we used an RNA polymerase derived from derived from T7 bacteriophage, a type of virus that infects bacteria, and called T7 RNA polymerase. T7 was added to the 5’ end of both of the THREAD and both of the dynamin related protein 1 primers that were used in the PCR. With the final double-stranded RNA we were able to perform RNA interference which is a cellular mechanism that uses the gene's own DNA sequence of gene to turn it off. This all works together because the elongation phase of transcription is often a critical crossroad for regulating gene expression and a strong initiation of RNA synthesis. RNA polymerase II can pause, arrest, pass through terminator sequences, or terminate transcription. The varying development of RNA pol II prior to entering productive elongation is controlled by the action of both negative and positive transcription elongation factors. For example, an experiment done Modulating HIV-1 replication by RNA interference used RNA interference to address whether hSpt5 is required for Tat transactivation. RNAi is an exceptionally efficient process in which double-stranded RNA activates sequence-specific deterioration of homologous mRNA in animal and plant cells. They found that in mammalian cells and RNAi can interfere with duplexes and a few dsRNA molecules. They also found that this accomplished a continuously inactivate transcribed target of mRNA for an observable period of time. Recently RNAi has been used to successfully knockdown the expression of a number of HIV genes (Yueh-Hsin). Apoptosis has a number of crucial roles in a variety of cellular and developmental processes. Programmed cell death, or apoptosis, is a genetically determined process by which a cell self-destructs for the benefit of the whole organism. In Drosophila, a key component in apoptosis is the inhibitor of apoptosis protein 1 (DIAP1, or Thread). Drosophila has been used as an admirable model to study apoptosis because of its advantages in genetic manipulation (Huang). The other gene of interest mention above, Drp-1 is also related to apoptosis. Drp-1 Not only regulates mitochondrial fission in normal cells, but mediates mitochondrial fragmentation during programmed cell death (Goyal). In these labs we amplified Thread1 and dynamin related protein 1 cDNA, and used RNA interference to observe the silencing of both apoptosis related proteins. How original hypothesis was for the amount of Drosophila cells to decrease after exposing them to the purified RNAi inhibitor. Without the access to apoptosis proteins, Drosophila cells were expected to die off because they could not kill off any bad cells,