• Polymerase Chain Reaction (PCR) is the technique used by biochemists and molecular biologists to amplify one piece of DNA and generate millions of copies. At TSoG we have utilized PCR on several occasions. As part of SEADD 2017 we amplified the 16s rRNA gene of soil bacteria isolated from the Girl Scouts Camp Whispering Cedars. The 16s rRNA gene is the DNA sequence that encodes part of the small ribosomal subunit in prokaryotes, because it is highly conserved between species of bacteria is used to classify microbes. During summer 2017 used PCR at the TSoG lab for Mentorship students to analyze their own genomic DNA. Student determined if they carried variations in a gene that confers the phenotypic ability to taste bitter flavors, like those found in broccoli. We found out several of our Copernicus and Kepler students carry the single nucleotide polymorphism (SNP) and are super tasters! We also used PCR in BIO1112, the Kepler level microbiology unit, this past fall. Our students used bioinformatics databases to design a multiplex PCR assay – an experiment where you amplify multiple pieces of DNA in one reaction – to test for the presence of the foodborne pathogen E. coli O157:H7 in unknown and environmental samples. They chose one of the genes that we tested for, collected their own samples to test, then carried out the PCR experiment, and analyzed the results via gel electrophoresis.


    PCR as we currently know it was developed by Karry B. Mullis in 1983 and he was awarded the Nobel Prize in chemistry in 1993 for his work. PCR has revolutionized the research and healthcare communities and is now a routine laboratory procedure. PCR has many applications. In healthcare it is used for screening and diagnosis of genetic diseases, diagnosing infectious diseases, and is what makes personalized medicine of diseases such as cancer possible. In research it is used in genetic engineering, analysis of gene expression, and identifying organisms like bacteria. Besides identifying bacteria, PCR is also how forensic scientists make DNA matches in criminal cases and how archeologists can analyze DNA in unearthed biological specimens.


    To carry out a PCR experiment you need a sample of DNA that contains the sequence you want to amplify, a DNA polymerase, two primers, deoxynucleotide triphosphates or dNTPS, a buffer, and magnesium or manganese ions. DNA polymerases are a family of enzymes that synthesize DNA; they form the DNA polymer from the building blocks of DNA, deoxyribonucleotide monomers. Taq polymerase is the most commonly used DNA polymerase in PCR. It is a thermostable (heat stable) enzyme that was isolated from a thermophilic (heat loving) bacteria found in hot springs and hydrothermal vents. It is used because it can survive the highest temperature needed in PCR, which is too hot for the DNA polymerases found in other organisms, like humans. Magnesium or Manganese are needed because they act as co-factors for the polymerase enzyme. DNA primers are small strands of DNA that are complimentary to the three prime ends of each strand of target DNA. Primers are used because DNA polymerases can only bind and elongate double stranded DNA and the double stranded target DNA will be separated into single strands during the PCR cycle. Once the target DNA has been separated into single strands, the primers will bind to their complimentary sequences on the target strands creating small double stranded segments which act as the starting points for elongation. dNTPs are deoxyribonucleotides (a nucleobase, sugar, and one phosphate) bound to an additional two phosphate groups – the T stands for triphosphate. During the reaction they lose two of those phosphate groups, which provides the chemical energy needed for polymerization. This leaves them with only one phosphate, now the deoxyribonucleotide monomers of DNA. All four types of deoxyribonucleotides - based on the nucleobases adenine, guanine, cytosine, and thymine - must be included in the reaction.


    PCR is carried out in a thermocycler, which is an instrument which heats and cools the reaction to the temperatures that are required for each step of the process. One PCR cycle consists of three repeated steps which occur at different temperatures. The first step in the cycle is the denaturation step, which is carried out at 94-96 °C. The high temperature causes the template DNA to melt, producing the single stranded DNA molecules. The second step is the annealing step, where the temperature is lowered to 50-68 °C. This lower temperature allows the primers to hybridize or bind to the complimentary strand of target DNA. The range for the annealing step is broad, because it depends on the primers being used and their melting temperatures. If the temperature is too high, the DNA will remain melted and not anneal at all; if it is too low, the primer may bind in the incorrect part of the DNA sequence leading to erroneous replication. The third step is elongation step, and is done at the optimum temperature for the polymerase being used. This is around 72 °C for Taq Polymerase. After each cycle the quantity of DNA will double, leading to exponential amplification of the target gene until you either run out of dNTPs, the enzyme loses activity, or the cycle is stopped.



    Although PCR is commonly used in laboratories and relatively inexpensive compared to some other laboratory experiments, the cost of PCR equipment can be prohibitively expensive for use in classroom settings. At TSoG we use PCR equipment developed by a small start-up founded by two Ph.D. scientists called MiniPCR. The MiniPCR systems are inexpensive, compact, and provide safe, all-in-one solutions to carrying out PCR and analyzing PCR products. Sebastian Kraves, a Ph.D. neuroscientist and co-founder of MiniPCR, talks about some of the awesome things that can be done with miniaturized PCR machines during a TEDtalk here. Besides offering great PCR solutions for classroom applications, miniPCR systems have been used to do PCR in space on the International Space Station (ISS) and on the ground in Sierra Leone during the Ebola outbreak. MiniPCR even hosts an annual Genes in Space contest for middle and high school students to design PCR experiments to be done in space on the ISS! The winning proposal will be performed in space and the winner(s) also get to take part in a host of other cool science experiences, like presenting their experiment at conferences and attending a space biology camp. Students grades 7-12 can participate individually or in teams of up to 4. Each proposal must be mentored by an adult, which I will be more than happy to do for anyone who would like participate! 2018 marks the fourth year for the Genes in Space competition. Last year, TSoG alum Jennifer Pearson was awarded an honorable mention. The 2018 Genes in Space competition opens up for submissions on January 16th and Experiment proposals are due by April 20th, 2018. You can find a video that tells more about MiniPCR as a company and a little about the competition here and full contest information here. Please ask me if you have any questions about PCR in general and/or if you would like to submit a proposal! I would really like to see multiple proposals submitted by our Copernicus, Kepler, and Newton students!


    Sincerely,
    Ms. Dark

  • Polymerase Chain Reaction (PCR) is the technique used by biochemists and molecular biologists to amplify one piece of DNA and generate millions of copies. At TSoG we have utilized PCR on several occasions. As part of SEADD 2017 we amplified the 16s rRNA gene of soil bacteria isolated from the Girl Scouts Camp Whispering Cedars. The 16s rRNA gene is the DNA sequence that encodes part of the small ribosomal subunit in prokaryotes, because it is highly conserved between species of bacteria is used to classify microbes. During summer 2017 used PCR at the TSoG lab for Mentorship students to analyze their own genomic DNA. Student determined if they carried variations in a gene that confers the phenotypic ability to taste bitter flavors, like those found in broccoli. We found out several of our Copernicus and Kepler students carry the single nucleotide polymorphism (SNP) and are super tasters! We also used PCR in BIO1112, the Kepler level microbiology unit, this past fall. Our students used bioinformatics databases to design a multiplex PCR assay – an experiment where you amplify multiple pieces of DNA in one reaction – to test for the presence of the foodborne pathogen E. coli O157:H7 in unknown and environmental samples. They chose one of the genes that we tested for, collected their own samples to test, then carried out the PCR experiment, and analyzed the results via gel electrophoresis.

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