Cohesion Phenomics Internship - Summer 2018 (McKenzi Sexton)
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Here is a PPT McKenzi made about her summer internship experience at Cohesion Phenomics with Dr. Sumy Joseph.
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Cohesion Phenomics Internship - Summer 2018 (McKenzi Sexton)
- 1. 2018 SUMMER INTERNSHIP McKenzi Sexton June 2018
- 2. Cohesion Phenomics Clinical genetics laboratory Spindale, NC test for gene disorders specifically related to cardiovascular diseases
- 3. How I Got Involved Flyer Application Essays Interview process
- 4. The Internship June 4th – June 21st Monday – Thursday, 10:00am to 4:00pm
- 5. Their Model • Observe • Equipment, safety, HIPAA, and housekeeping Week One • Be Observed • Factor V Leiden/Factor 2 and HFE allelic discrimination assays Week Two • Perform • FVL/F2, making gels, electrophoresis, and analyzing results Week Three
- 6. THE PROCESS
- 7. Isolation whole blood in purple-capped tubes color of lid corresponds to the anticoagulant used Purple: K2 EDTA, strong anticoagulant Isolate genomic DNA from whole blood Hood, PPE = engineering controls to avoid infection by blood borne pathogens
- 8. Isolation (cont.) Quantitate using a fluorometer to determine concentration and yield accurately detects very low concentrations of double-stranded DNA Isolation yields 2-4uL
- 9. PCR Polymerase Chain Reaction Exponentially amplifies a single copy of target DNA 1. Denaturation of template by heat 2. Annealing of primers to target sequence 3. Extension of primers by DNA polymerase PCR product = cut up pieces of target DNA and primer, which can be… 1. Run on a gel to determine base pair size and quality of product 2. Sequenced 3. Used in a digest/ allelic discrimination assay
- 10. 1. Gel Electrophoresis Method of separating segments of DNA by size Phosphate in DNA backbone has a negative charge Moves toward positive electrode Larger segments can’t pass through the pores in the gel as easily as smaller segments
- 11. Pore sizes in agarose gel vary depending on the gel’s purpose… Check quality of genomic DNA Determine sizes of PCR fragments, quality of product, and primer excess Separate cut sections of DNA after digest
- 12. 2. Sequencing PCR product is cleaned with Exosap gets rid of excess reagents and primers M13 tag is added Product is cleaned with BDX gets rid of leftover dyed nucleotides, excess reagents 3500 Genetic Analyzer
- 13. 3. Digest/Allelic Discrimination Used to identify disease-causing mutations Restriction enzymes added to PCR product DNA is cut at specific points where a mutation will be located Electrophoresed on 3% gel and imaged
- 15. Disorders Factor V Leiden ThrombophiliaFVL • Blood clotting disorder, poor anticoagulant response to APC, increased risk of VTE (DVT) • G-to-A substitution at nucleotide 1691 on F5 gene Factor 2 Prothrombin ThrombophiliaF2 • Blood clotting disorder, similar to FVL • G-to-A substitution at nucleotide 20210 on Prothrombin gene HaemochromatosisHFE • Inherited iron overload disorder • C282Y allele, H63D allele = G-to-A transition point mutation of the HFE (high iron) gene
- 16. Number of alleles affects clinical expression Heterozygotes (+/-) FVL: slightly increased (3- to 8-fold) risk for venous thrombosis F2: moderately increased prothrombin levels and an increased (3- to 8- fold) risk for venous thrombosis HFE: C282Y: no increased risk of iron overload H63D: no increased risk Both: slightly increased risk Homozygotes (-/-) FVL: much greater (18- to 80-fold) thrombotic risk F2: high prothrombin levels and a greatly increased risk for venous thrombosis HFE: C282Y: greatly increased risk H63D: very slightly increased risk
- 17. My Results – FVL/F2 Being Observed 18-0001 FV: +/- CP-170556: +/- ctrl 18-0002 F2: +/+ CP-150419 F2: +/- ctrl
- 18. My Results – HFE Being Observed 18-0003 CY: +/- HFE-GGC-3: +/- ctrl* 18-0004 HD: -/- HFE-GGC-8: +/- ctrl *pipette error
- 19. My Results – FVL/F2 Performed
- 20. My Results – FVL/F2 Performed 18-0005 FV: +/- CP-170556-FV: +/- ctrl 18-0006 F2: +/+ CP-150419-F2*: +/- ctrl *pipette error
- 21. Additional Things I Learned
- 22. Equipment Pipettes Mouth pipetting Gel apparatus UVP Thermal cycler Laboratory grade dishwasher Water filtration system
- 23. Temperature Data Responsible for taking A.M. and P.M. temperatures and humidity Ranges ensure proper equipment function Root cause analysis Positive pressure and reduced airflow in Isolation room and PCR room
- 24. Temperature Data – Isolation Room 60 62 64 66 68 70 72 74 Isolation Room Temperatures Acceptable Range: 59°F - 104°F Acceptable Range A.M. P.M. 50% 55% 60% 65% 70% 75% 80% Isolation Room Humidity Acceptable Range: 20% - 75% Acceptable Range A.M P.M.
- 25. Temperature Data – PCR Room 65 66 67 68 69 70 71 72 73 74 75 PCR Room Temperatures Acceptable Range: 39.2°F-104°F Acceptable Range A.M. P.M. 10% 20% 30% 40% 50% 60% 70% 80% PCR Room Humidity Acceptable Range: 20% - 75% Acceptable Range A.M. P.M.
- 26. Temperature Data – Main Lab 10% 20% 30% 40% 50% 60% 70% 80% 90% Main Lab Humidity Acceptable Range: 20% - 80% Acceptable Range A.M. P.M. 65 66 67 68 69 70 71 72 73 74 75 Main Lab Temperatures Acceptable Range: 59°F - 86°F Acceptable Range A.M. P.M.
- 27. Resolution: temperatures will be taken in P.M. after a full day of work for more accurate representation Temperature Data
- 28. Conclusion
- 29. What I brought from school Basic lab skills Micropipetting (Genetics and Microbiology) Gel electrophoresis (Genetics and Microbiology) Aseptic technique (Microbiology) Autoclaving (Microbiology TA)
- 30. Pre-Internship Goals Develop new laboratory skills and improve current ones Gain insight into working in a lab Gain experience in my field of interest
- 31. Post-Internship Goals Confirmed I would enjoy working in a lab Also allowed me to develop important field-related skills, familiarity with equipment Use and expand on acquired skills In GWU labs, in the workplace Use this experience to hopefully secure more opportunities to learn about or work in the field
- 32. Thank You!
Editor's Notes
- Good afternoon! My name is McKenzi Sexton and today I’ll be talking to you about my internship with Cohesion Phenomics.
- Cohesion Phenomics is a small, high complexity clinical genetics laboratory located in nearby Spindale, North Carolina. They test for and analyze gene disorders in patients, specifically related to cardiovascular diseases.
- I became interested in this opportunity back in February after Mrs. Manahan encouraged my Genetics class to apply for it. The timing was basically perfect – I’ve been interested in the field of genetics and had been looking into internships that were genetics adjacent but not quite right throughout the week leading up to that day. I took a look at the flyers pinned up in the hallway immediately after and picked up an application from Mrs. Manahan’s door on my way back to my dorm. The paper application involved listing any relevant coursework (with one of the requirements being the completion of both Gen Chems, General Bio, and Micro), your GPA (which needed to be above 3.0), and any relevant field experience. In addition to this, they asked for the submission of two short essays, one being your career goals and the other being your interest in healthcare (which I didn’t necessarily have and it still worked out). I was notified that I had received an interview a couple weeks later and went in to be interviewed, meet the staff, and receive a tour of the lab in early May.
- I interned at the lab Monday through Thursday 10:00am to 4:00pm for three weeks in June. This internship followed the model Cohesion Phenomics uses to train their new lab technologists:
- Observe, Be Observed, Perform. It’s a solid model that helped really cement what I learned throughout the internship. The first week involved orientation, safety, learning about the equipment, practicing using the micropipettes, and largely watching the lab tech complete procedures and other work in the lab. The second week still involved some training, but it also involved me getting my “hands wet” for the first time making gels for gel electrophoresis and performing two allelic discrimination assays under her supervision. During the third week, I still made gels and did a third allelic discrimination assay, and I also made running buffer for gel electrophoresis, did some housekeeping, imaged the two gels that I had run, and analyzed them for the presence of alleles that were associated with the genetic heart disease we were testing for, all under limited or no supervision.
- What was really cool about this internship was that I got to see the entire process of the testing they do at Cohesion Phenomics, from getting in the actual blood samples all the way through to the results. Because the lab is small, the lab tech I shadowed was responsible for the entire process as opposed to just one or two steps. The highlighted pathway is the one the internship provides the most experience with.
- The first step is always isolation. Whole blood is delivered in purple-capped tubes. The purple color designates the use of the strong anticoagulant EDTA. EDTA tubes are preferred by most molecular genetics laboratories for molecular genetic studies. Genomic DNA can be isolated from whole blood or spun in a centrifuge to separate out the buffy coat layer and isolated from that. I got to watch an isolation from whole blood, which involved first lysing the anucleate red blood cells. The tube is pun down in a centrifuge to “pellet,” or clump together, the remaining white blood cells. The lysed red blood cells and buffer are removed and the pellet is resuspended in GB buffer. The hood is an engineering control used to avoid infection by blood borne pathogens when working with blood, but at this point the sample is no longer hazardous and can be worked with on the bench. A binding solution for the DNA is created by adding ethanol. Everything is put in a spin column, which contains thin layers of glass that catch DNA and a hole in the bottom to allow anything else to run out when spun in the centrifuge. After a couple more steps where the DNA is washed and eluted, the DNA is then purified.
- A fluorometer is used to determine the concentration and yield of the purified DNA. Fluorometers use fluorometric detectors that emit fluorescence when bound to target molecules, allowing the machine to accurately detect very low concentrations of double-stranded DNA. Typically, isolation gives you between 2 and 4 micrograms, or one one millionth of a gram, of genomic DNA. After quantitation, the samples are ready to undergo PCR.
- PCR stands for Polymerase Chain Reaction and is a way to pick out a specific target section of DNA and exponentially amplify it. The DNA from isolation is used along with water, primers, and DNA polymerase to make the PCR mix. Different patient samples and their appropriate primers are mixed in individual tubes and pipetting into wells on a plate, which is covered and placed in a thermal cycler, which automates the entire heating and cooling process. PCR works by first heating the samples to denature the template DNA and separate the two strands. The samples are then cooled enough to allow the primers, or short pieces of DNA that are used to bind to the specific segment of DNA that we want, to anneal to the target sequence of DNA. The DNA polymerase is an enzyme that creates DNA molecules by adding nucleotides to the template DNA strand and is used to extend the primers and ultimately make double the original number of double-stranded DNA molecules. The product of PCR is cut up pieces of target DNA and primer, which can be either 1) run on a gel to determine base pair size and robustness, 2) sequenced, or 3) used in an allelic discrimination assay, also referred to as a digest.
- Gel electrophoresis is a method of separating segments of DNA by size. This works because the phosphate in the sugar-phosphate backbone of DNA has a negative charge. The electrophoresis apparatus runs an electrical current through an agarose gel submerged in running buffer to move the DNA from the wells in the gel near the negative electrode toward the end of the gel near the positive electrode. As the segments of DNA move, larger segments can’t pass through the pores in the gel as easily as the smaller segments, so the larger DNA segments move more slowly through the gel than the smaller ones. The pore sizes in the agarose gel can vary depending on the purpose of the gel.
- Pore sizes in agarose gel vary depending on the gel’s purpose. The smaller the number, the larger the pores in the gel are, and vice versa. 0.5%: The quality of the genomic DNA obtained after isolation can be determined with a 0.5% gel check. Genomic DNA is large with a high molecular weight and the 0.5% gel has the largest pores. Electrophoresing genomic DNA allows us to check the concentration, with bolder, brighter bands meaning the higher the concentration. The stability can also be determined, with crowning meaning the DNA is unstable, and because DNA is very fragile and can be sheered by pipetting, the incidence of sheering can be seen by a second band appearing far below the actual band. 1.5%: The 1.5% gel has smaller pores and is used to determine the size of PCR fragments in base pairs, which was one of the uses for PCR products like previously mentioned. It also helps to show how well the PCR reaction worked by showing the product robustness in the same way as with the 0.5% gel check. 3%: The 3.0% gel is used to separate out cut sections of DNA after a digest. This gel has the smallest pores and produces the finest image.
- The second use for a PCR product is sequencing. In this process, the PCR product is cleaned with Exosap, which gets rid of leftover magnesium and excess reagents. The M13 tag is added to let polymerase know where to attach to start sequencing/replication. Sequencing is done with the 3500 Genetic Analyzer, which is a sequencing instrument that works kind of like electrophoresis, but determines nucleotide peaks on a computer instead of sizes of DNA fragments in the form of bands. Instead of agarose gel, pop is used, which is comparable to a 9% gel and is the finest pore size used in this lab, and instead of running buffer, anode and cathode buffer is used.
- The third use for a PCR product, and the one I saw the most, was the allelic discrimination assay, which is used to identify disease-causing mutations. After PCR, the appropriate buffer and restriction enzymes are added and the digest mix is put back into the thermal cycler to cut the DNA at specific points where we know a mutation will be located. After the digest is complete, loading dye is added to each sample before the samples are loaded into a 3% gel, as previously mentioned. The gels are then imaged and the results are interpreted.
- This allelic discrimination assay was the procedure I was specifically prepared to do. Due to safety reasons involving the risk of contact with blood borne pathogens, I was not allowed to isolated any samples from blood, but I was able to do each of the rest of the steps to eventually identify disease-causing mutations in DNA.
- The diseases in particular are two blood clotting disorders and one iron overload disorder. Factor V Leiden Thrombophilia and Factor 2 Prothrombin Thrombophilia are two fairly similar blood clotting disorders that involve a heightened risk of venous thromboembolisms, or blood clots, particularly deep vein thrombosis in the legs. Both result from G-to-A substitutions at specific nucleotides in their respective genes. FVL results from the mutation at nucleotide 1691 on the factor 5 gene and F2 results from the mutation at nucleotide 20210 on the prothrombin gene. Haemochromatosis, or inherited iron overload disorder, involves excessive dietary iron uptake which accumulates in organs and disrupts their normal function. This is the result of another point mutation in the HFE (standing for high iron) gene. This can occur at two different locations, resulting in a C282Y allele or a H63D allele.
- The number of the mutant alleles that are present affects the clinical expression of these disorders. Heterozygotes have a higher risk for the effects of the disease compared to those with two normal alleles, but homozygotes have much higher risks. In FVL, heterozygotes have a 3- to 8-fold increased risk for venous thrombosis, while homozygotes have an 18- to 80-fold increased risk. In F2, heterozygotes have moderately increased prothrombin levels and a slightly increased risk for venous thrombosis much like FVL heterozygotes, while homozygotes have high prothrombin levels and a greatly increased risk for venous thrombosis. And in HFE, those heterozygous for the C282Y allele or the H63D allele have no increased risk of iron overload, but those that are heterozygous for both alleles have a slightly increased risk. Meanwhile, those homozygous for the C282Y allele have a greatly increased risk for iron overload and those homozygous for the H63D allele have a very slightly increased risk.
- During Week 2, when I was replicating the procedures I had watched under the lab tech’s supervision, I got to amplify, digest, and run the DNA on a gel and then image and analyze the results for both the FVL/F2 and the HFE assays. When loading a gel, the wells are set up with a no DNA control first, the patient samples, and the heterozygous control DNA. A 100bp ladder and a 25bp ladder are also added in order to determine the sizes of the DNA fragments. This table allows us to determine whether the sample has two normal alleles, are homozygous for a mutation, or are heterozygous for that mutation by using the known sizes of the fragments when cut. In this case, sample 1 was heterozygous for the FVL allele and sample 2 was homozygous for the non-mutated prothrombin allele.
- In the HFE allelic discrimination that was run on the gel at the same time, sample 3 was heterozygous for the C282Y allele, which was determined despite the heterozygous control not showing up due to pipette error. Sample 4 was found to by homozygous for the H63D allele.
- In Week 3, I performed a FVL/F2 allelic discrimination on my own. Once again, I made a pipette error that resulted in the heterozygous control, this time for the F2 test, not appear. Fortunately, the results could still be determine.
- It was found that sample 5 was heterozygous for the FVL allele and that sample 6 was homozygous for the non-mutated prothrombin allele.
- Aside from the allelic discrimination assays, there were many other aspects of labwork I got experience with.
- In Week 1, I had to go through the equipment manuals for every piece of equipment that I would be using. Some of these I have already discussed, but have now included pictures for. Here at Gardner-Webb, we use micropipettes in some labs, but I became really familiar with using them during this internship. It was the first time I had ever seen, much less used, a multi-channel pipette and I also learned about mouth pipetting, which is where apparently scientists would aspirate liquids into glass pipettes using their mouths as opposed to the bulbs we use in Organic. Here is a picture of the medium-sized gel apparatus, which is the one that I used most often, and here are the electrodes where the DNA moves from the negative black electrode to the positive red one. Here is a picture of the Biodoc-it gel imaging system, in which you put your gel in this dark room and use this screen to adjust the image. The last image is of the Veriti Thermal Cycler, which is the machine that does nearly the entire PCR process. This cover lifts up and your plate of samples sits inside, where it is heated from both sides.
- The temperature and humidity of the various rooms and appliances at Cohesion Phenomics must be monitored daily to ensure no testing occurs outside of the normal range, which is set in place according to the equipment manuals. Because the isolation and PCR rooms must not be contaminated in any way, they are under positive pressure, which means there is no air coming from the main lab, air is only pumped into the room by ceiling vents as needed. Due to this reduced airflow, problems developed especially with humidity. I was responsible for taking temperatures of each room, frige, and freezer upon arriving in the morning and before leaving in the evening. I have made a couple graphs showing the change in temperature and humidity at the two times for the isolation room, the PCR room, and the main lab.
- In the isolation room, temperatures remained within range for the duration of my internship, though there were some issues with the humidity some mornings. This is due in part to the positive pressure of the room, but also is likely due to the room’s location. This room borders the entrance room, which is small with an exterior wall composed almost entirely of windows. This room allows a lot of hot sunlight to beat down inside, raising the room’s temperature and likely having some influence on the conditions of the isolation room.
- The same was true for the PCR room, though here the humidity never increased beyond the acceptable level. In order to solve this problem in both the isolation and the PCR rooms, the doors were left cracked for a couple hours, which is the reason why there is such a significant difference in the A.M. and P.M. data for these days.
- The data for the main lab was largely consistent, though it was affected by the high humidity as well.
- This data was used to decide that daily temperatures should be taken in the afternoon. The conditions of each room were always warmer and more humid in the mornings than in the afternoon, due to inconsistencies with the air conditioning unit. Taking temperatures in the afternoon should allow for a more accurate representation of the conditions worked in.
- To conclude this presentation, I would like to touch on what I hoped to gain during this internship and how I hope to use my experience at Cohesion Phenomics in the future.
- Coming into this internship, I had had only brief hands-on experience with some of the tools used most frequently at Cohesion Phenomics. Micropipetting and gel electrophoresis apparatuses are occasionally used in Genetics and Microbiology. Aseptic technique was also a focus of Microbiology, and it is of the utmost importance in a clinical genetics lab. My work as a Microbiology teaching assistant had also allowed me to briefly see and use the autoclave at the end of lab days.
- Going into this, I hoped to really develop and improve the lab skills I had been taught at Gardner-Webb. I also was ready and hoping to learn new skills I would be able to apply in lab settings. My other goals for this internship were to get a feel for what working in a lab would actually be like, because I already felt like I would enjoy a career involving labwork but had no way of knowing, as well as to gain experience specific to genetics.
- I ended up thoroughly enjoying my experiencing and confirming myself that I would enjoy labwork long-term. I also did develop those pre-existing lab skills well beyond what I was expecting, receiving many invaluable tips and tricks. I realized that college science labs teach you to use a large variety of tools, but only barely. I didn’t realize how much I had to learn until I was learning it. I would love to use and expand on the skills I’ve gained during this internship, hopefully by finding a way to use them here at Gardner-Webb and definitely at some point in the workplace. I also hope to use my experience at Cohesion Phenomics to find ways to secure more opportunities to learn about the field of genetics.
- All in all, this internship at Cohesion Phenomics showed me what working in a lab and working in genetics might look like, prepared me for actual fulfilling work using a powerful model, and left me with new and improved skills and knowledge and a valuable experience. Thank you for listening.