MB&B/BIO 181 | Polymerase Chain Reaction (PCR)
Name: Teresa Naval
INTRODUCTION
Scientifically, why is the study of Alu insertions interesting? (10 pts) Alu insertions are biological tools that can trace human history, evolution, and migration. In particular, Alu is often used to understand prehistory because all primates (including humans) with Alu insertions at specific locations can be traced to common ancestors [3].
Alu is sometimes referred to as a “jumping gene” because it copies itself to shift to new locations. While it is technically not a gene, the way Alu insertions are inherited (i.e. passed down from parents to offspring) is much like that of a gene. It is not a gene, however, because Alu does not code for any protein. It
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Batzer and Deininger (2002) even mention that “Alu insertions account for ~0.1% of all human genetic disorders (p. 374)” including some forms of cancer and diabetes. Alu, then, allows researchers to create a biological map of human activity.
What are the objectives of this lab? (5 pts) Using polymerase chain reaction (PRC), this lab focuses on the amplification of DNA extracted from cheek cells. The experiment aims to discover the presence or absence of the human Alu insertion in the PV92 locus on chromosome 16. It aims to enable the students to understand how PCR works, and how it is used to understand DNA fragments.
Another objective of this lab is to understand the implications of Alu insertions in human evolution, and to examine some of the genomic variations within the human species using a sample population in the laboratory. METHODS
What steps are involved in carrying out PCR and biologically, what happens during each step? Why did we use PCR? (15 pts)
A. Preparing the Cheek Cells (Pre-PCR) The process of isolating cheek cells began with rinsing the inside of the mouth with 0.90% saline solution. A micropipette was used to transfer 1,500 uL of the solution into a tube, which was then placed in a microcentrifuge to spin at 8000 rpm for 90 seconds. The centrifuge made sure that the contents of the solution were segregated by weight, with the DNA precipitate (cell pellet) settling at the bottom. The supernatant was removed, and
20 ul of DNA was added to 20ul of Master Mix. The Master Mix contained primers, dNTPs, Mg2+, Taq DNA polymerase, and yellow dye. Both the DNA and Master Mix were mixed with the micropipette. The DNA was then put into the thermal cycler containing 40 cycles of PCR amplification, amounting to 3.5 hours of amplification.
(PCR), which isolates small fragments of DNA that have a high degree of variability from
Each 1.5 ml microcentrifude tube containing 50 µg myceliums, 50 µl of Lysozyme, 450 µl of Lysis solution and 3 µl of RNase A was kept for hour at 37 0C on water bath, vortex several times in duration. Then, addition 250 µl of 2% SDS, gently invert before addtion 250 µl of mixing Phenol- Chloroform- Isoamylclcohol ratio is 25:24:1, vortex and centrifuge at 12,000 rpm in 10 minutes then transfer supernatant on up layer to a new 1.5 ml microcentrifuge and repeat that step again before the next step to completely remove unexpected components. Subsequently, 0.1X of 3M Sodium acetate pH 4.8 was supplemented,
Then 5 µL of each PCR sample was loaded onto a 2% agarose gel in the following order: Bioneer 25/100bp mixed DNA ladder, WT CCR5 plasmid, ∆32 CCR5 plasmid, cheek DNA, negative control. The gel was allowed to develop.
The process of PCR has 3 steps that repeat in cycles shown in image 1. The first one is the separation of the two strands of the DNA molecule. This happens by the initial material being heated to 95 °C. Every DNA
Alu sequencing is an important part of the human genome process. It helps determine our genotypes and alleles. These Alu elements also help create a genetic diversity in the human genome. Once an Alu inserts itself at a chromosomal locus, it can copy itself for transpositions. Each Alu element has an internal promoter for RNA polymerase III that is needed to initiate transcription. The PV92 genetic system has two alleles that indicates the presence (+) or the absence (-) of the Alu element on each of the paired chromosomes. This will then result in three PV92 genotypes: ++, +-, or --. The positive and negative alleles can be separated by its size using the gel electrophoresis. While we are observing an Alu element in the PV92 region of chromosome
The polymerase chain reaction or PCR for short can be used to create many copies of DNA. This allows the DNA to then be visualized using a dye like ethidium bromide after gel electrophoresis. The process has been refined over the years, however the basic steps are similar.
DNA is mostly found in the nucleus of nearly every cell in the human body, it contains the biological instructions that make us unique. Located on the genome at different locations are short, identifiable sequences known as Alu insertion polymorphisms. The application of Alu elements has recently become used in forensic identification and paternity testing. Alu elements are found in nearly one million copies per haploid gene which approximately 5 – 10% of the human genome. The Alu elements can be found bordering genes or gathered with other interspersed repeated sequences (Primrose 1998).
DNA is isolated from the blood sample and amplified through the polymerase chain reaction (PCR), a technique for rapidly producing,
Genomic research is extremely important and beneficial for understanding and treating diseases. The research on this topic grows each and every day as scientists continue to learn new things about our genomes and how they can be helpful in our everyday lives. All humans genomes are essentially the same in all people and that genetic differences make up about one tenth of a percent of our DNA. Those genetic differences can have a profound impact on health problems encountered by individuals (Roche, 2016). As genomic research grows, people will soon be able to know their entire karyotype and be able
Like in any biological field we used a Polymerase Chain Reaction (or PCR for short), which is used to amplify “specific DNA
To learn more about the genetic variation within out class, we chose two loci to examine: the TAS2R38 gene located on chromosome 7 and the PV92 Alu
PCR tubes were ready for purification when a single DNA band was present per lane on the agarose gel. If multiple bands were obtained in a single lane the PCR process and gel visualization process was repeated however, the denaturation temperature was run at a higher temperature to prevent non-specific binding of the primers to the DNA template. The purification process used an Invitrogen PCR clean-up kit. 5mL of charge switch beads
Reverse transcriptase polymerase chain reaction, also known as RT-PCR, has been recognized as a reliable, accurate, and sensitive method for quantifying gene transcription. Polymerase chain reaction, also known as PCR, is considered an essential tool in molecular biology that allows for the amplification of nucleic acid sequences. Specifically, the three main consecutively repeating steps in PCR are denaturation, annealing, and elongation. If the reaction runs with 100% efficiency, there will be a two-fold increase in target amplicons after each cycle of PCR. Therefore, with n cycles of the reaction, the copy number of the target sequences will be 2n. However, one of the main disadvantages of conventional PCR, also called end-point PCR, is that the results of amplification can only be visualized after all the cycles of amplification are complete (Nestorov 326).
PCR is biochemical research technique developed in 1985 by Kary Mullins. It is based on the principle of amplifying DNA through a series of cycling reactions with varying temperatures. PCR uses the ability of thermostable polymerase enzymes to synthesize a new complementary DNA strand to a template strand.