Sanger Sequencing: Polymerase Chain Analysis

There were several steps used to acquire the colony necessary for the PCR. First a student forearm was swabbed using a cotton swab, the cells were then placed in an agar plate. DNA was then extracted from the cultured bacteria by using a technique to lyse the cells and solubilize the DNA, then enzymes were used to remove contaminating proteins. The DNA extraction consisted of a lysis buffer that contained high concentrations of salt for denaturing. Binding with the use of ethanol and a washing step to purify the DNA. The final step for the DNA extraction was elution where the pure DNA was release. Proceeding the extraction of DNA the results of the 16s gene amplification were examined through gel electrophoresis it was analyzed by estimating the size of the PCR bands with marker bands. After measuring the success of the extraction, a technique called TA cloning was started. Cloning of PCR products was done by using partially purified amplified products with

The Individual vs The system The films Girl, Interrupted (1999) and One flew over the Cuckoo's Nest (1975) convey the key themes of the balance between individual and societal control and how the individual navigates its constraints within 1960's psychiatric institutes. In both films, conformity and restriction drive the narrative into a story of resilience and rebellion against the systems control. This demonstrates the conflict between the individual and the system. In the film Girl, Interrupted, a young girl named Suzanna is placed into a psychiatric institution after an attempted suicide. When Suzanna speaks to a therapist about this, he relies on stereotypical questions such as "Do you have a boyfriend?"

Two different DNA sequencing techniques were used in this study. Sanger sequencing is a form of DNA sequencing in which the target DNA is copied multiple times in fragments of varying lengths. The ends of the fragments are marked with fluorescent chain terminator nucleotides that indicate the end of the fragments. These fragments would then be aligned according to any overlapping segments that they shared. This allowed larger regions of DNA to be sequenced via capillary gel electrophoresis (Khan Academy, n.d.). This sequencing method was used to sequence the human genome, but has since then become one of the more expensive and less efficient way to sequence.

The primary purpose is to identify a genetic marker or study the function of a specific gene. There are three steps involved in this process which are as follows: denaturation, annealing and elongation. Denaturation involves heating the DNA to agitate the hydrogen bonds, and annealing allows the temperature to be lowered so that the primers can be “annealed” to the single-stranded DNA template. The last step requires DNA polymerase to synthesize a new strand of DNA that is complementary to the RNA strand in the 5’ to 3’ direction (Amplifying DNA: The Polymerase Chain Reaction, 2016). The forward and reverse primers are needed to start the replication process by providing the appropriate nucleotides to the new strand. On the contrary, sanger sequencing makes copies of a target DNA, and the the DNA strand that will be sequenced is separated into two strands, so they can be copied through chemically altered bases. The altered bases cause the process of copying to terminate each time a particular letter is added to the growing DNA chain, which happens to all four bases until the fragments are put together to reveal the original sequence of the original DNA. The aforementioned processes are thoroughly explained to give an overview of the steps involved in providing the end products of the experiment, so an individual can manually decipher

Sacrifice Renee Rocco, author of Twisted, proclaims, “A hero will sacrifice the person they love to save the world, but a villain will sacrifice the world to save the person they love.” Refugee is an exquisite novel written by Alan Gratz, that revolves around three preeminent characters from three separate time periods: Nazi Germany, Cuba in the 90’s, and modern-day Syria. It follows Josef Landau, a German Jew attempting to escape from Germany to Cuba; Isabel Fernandez, attempting to escape Cuba’s poverty, corruption, and hunger crisis, to the United States; and Mahmoud Bishara, whose home gets destroyed by a missile, and is forced to flee. He and his family attempt to seek asylum in Germany. Their journeys are dictated by sacrifice and limitations,

In this experiment, host NM554, a particular strain of E. coli, was used to cultivate human genes (Dolf, 2013). Through the use of cosmids, plasmids that carry the cos gene, DNA fragments were introduced into the E. coli and packaged into phage particles (McClean, 1998). Pst I is a restriction endonuclease, an enzyme that cuts DNA at restriction sites (Restriction endonuclease). The Pst I digest of human DNA in this study produced the DNA fragments that were examined. The dideoxynucleotide chain-reaction procedure, also known as Sanger sequencing, is the process of lengthening DNA using DNA polymerase to add on deoxynucleotides until a dideoxynucleotide is added on randomly (Rogers). Fluorescence in situ hybridization (represented by the acronym

Because human DNA is being used, participants must fill out a consent form. Gel electrophoresis will determine the success of polymerase chain reactions. Some of the DNA will be shipped to Cornell University for the Biotechnology Resource Center to sequence. Using the NCBI's BLAST search engine, sequences will be compared to those in NCBI's database to see if the right region was amplified. DNA sequence analysis will help identify SNPs and amino acid present in

We used Big Dye which contained DNA polymerase, nucleotides, and dideoxynucleotides which were fluorescently labeled. The sequencing reaction will produce DNA fragments ranging from about 30 to 500 nucleotides (Holbrook and Leicht, 2017). To determine how much of our PCR DNA should be added to the sequencing reaction we observed the brightness of our 500 base pair band compared to the Size Standard. Our band was about twice as bright so we knew that our sample contained 13.3 ng/ul of 6 / 10 DNA. To make our dilution of our PCR product we combined 2ul of the DNA product to 14ul of water.

The principle of PacBio sequencing is easily understood, which captures sequence information during the replication process of the target DNA molecule. The template, called a SMRTbell, is a closed, single-stranded circular DNA that is created by ligating hairpin adaptors to both ends of a target double-stranded DNA (dsDNA) molecule [2]. When a sample of SMRTbell is loaded to a chip called a SMRT cell, a SMRTbell diffuses into a sequencing unit called a zero-mode wave guide (ZMW), which provides the smallest available volume for light detection [3]. In each ZMW, a single DNA polymerase is immobilized at the bottom, which can bind to either hairpin adaptor of the SMRTbell and start the replication. Four fluorescent labeled nucleotides, which generate distinct emission spectrums, are added to the SMRT cell. As a

The TARGet website allowed us to determine the Flanking sequence and DNA sequence of the homologous, which we then transferred onto Benchling. The Benchling website helped us determine the introns and exons present in our DNA sequence, as presented in Figure 3. When using benchling we also created primers that cover as various exons and introns as possible. Once we determined the forward and reverse primers that were to be used in our sequence, we ordered them and once received added resuspension buffer according to the instructions on the paper. We then diluted the forward and reverse primers and created yet another PCR table as shown in Figure 4 and once again created another master mix and ran electrophoresis on them. Our results can be seen in Figure 5. We ran the same dilutions a second time to clarify our results for the first PCR reaction. Those results can be seen in Figure

One test utilized in single-gene testing (1). Single-gene testing usually utilize Sanger sequencing, followed by real time-PCR in order to detect deletions or duplications in the DNA. Sanger Sequencing is the process DNA replication done in vitro with the use of chain-terminating dideoxynuleotides alongside DNA polymerase. Real time-PCR is the process of amplifying a single copy of DNA to point of gaining millions of copies of the DNA.

This method, as well as the Maxam and Gilbert method, for sequencing DNA are transforming the world of science, medicine and the views of people around the world.

Whereas the reaction products are not specific the SYBR green dye added to the reaction mixture to quantitatively indicate the amount of dsDNA in the reaction mixture by providing amplification plot and melt curve as well as the melt temperature plot that used in may offers useful information about the sensitivity of the DNA template in the sample (Giglio, Monis and Saint, 2003).

Like other next-generation sequencing methods, three general steps are required for exome sequencing: library creation, sequencing, and data analysis. The size and quality of tested samples are screened before establishing the library. In library creating, DNA molecules are fragmented into a suitable size and fused with platform-specific adapters. After size selection step free adapters elimination, PCR is performed to select for molecules containing adapters at both ends and to generate sufficient quantities for sequencing2. The sequencing is performed in specific machine such as Illumina Hi-Seq Platform, the working principles were discussed in detail in Topic Three, however, in exome sequencing, only exomes are selected, amplified and

To get high-quality clean reads, the assessment of sequencing quality and read processing were performed by fastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). The low-quality reads were removed using the same filtering criteria as described previously []. The clean reads were aligned on the F. vesca reference genome [1] using HISAT2 using default parameters [2]. Stringtie with higher accuracy and precision was used to generate transcript assembly [2]. Meanwhile, all unmapped reads derived from the above libraries were de novo assembled using the Trinity under default parameters to obtain comprehensive