BME100 s2018:Group7 W1030 L4

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Name: Dragos Baron
Name: Mery Montenegro
Name: Lauren Lossner
Name: Zachary Vance

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reac on mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s
  • DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA
  • A strip of empty PCR tubes
  • Disposable pipettes
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR Machine


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G# + Positive control none
G# - Negative control none
G# 1-1 Patient 1, replicate 1 46793
G# 1-2 Patient 1, replicate 2 46793
G# 1-3 Patient 1, replicate 3 46793
G# 2-1 Patient 2, replicate 1 31082
G# 2-2 Patient 2, replicate 2 31082
G# 2-3 Patient 2, replicate 3 31082


DNA Sample Set-up Procedure

  1. Label each PCR tube based on the specific DNA samples
  2. Transport the positive control reaction sample to the PCR tube labeled G + using one of the micropipettes
  3. Using a new pipette, transport the negative control reaction sample to the PCR tube labeled G -
  4. Transport the Patient 1 DNA sample to the tubes labeled G 1-1, G 1-2, and G 1-3
  5. Transport the Patient 2 DNA sample to the tubes labeled G 2-1, G 2-2, and G 2-3
  6. Transport PCR reaction mix to each PCR tube with DNA in it using a new micropipette for each tube
  7. Put the PCR tubes in the OpenPCR machine
  8. Start the machine and allow 25 cycles to occur


OpenPCR program

HEATED LID: 100°C

INITIAL STEP: 95°C for 2 minutes

NUMBER OF CYCLES: 25

  • Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 seconds

FINAL STEP: 72°C for 2 minutes

FINAL HOLD: 4°C



Research and Development

PCR - The Underlying Technology

Function of Each Component of a PCR Reaction

Template DNA replicates itself into 100 billion identical copies.Primers attach to sites on the DNA strands that are at either end of the segment that is being copied. They are important for replicating specific DNA sequences since there is little chance of coming in contact with the wrong sites. Taq Polymerase are polymerase molecules that act like tiny machines that read the DNA code and then attach matching nucleotides to create DNA copies. They are elongated. The deoxyribonucleotides (dNTP’s) have to be added in order to elongate the nucleotides.The DNA polymerase grabs nucleotides that are floating around in the liquid around it and attaches them to the end of a primer.


Steps of Thermal Cycling

At 95 degrees celsius, the DNA double helix begins to separate, which then creates two single-stranded DNA molecules. The thermal cycler cools down to 50 degrees Celsius, causing the single strand DNA molecules to pair up but there are too many primer sequences than DNA strands. The primers overcrowd and lock onto their target before strands can rejoin. The thermal cycler warms up to 72 degrees celsius, activating the DNA polymerase. When DNA polymerase locates an attached primer with a single DNA strand, it begins to add a complementary nucleotide. It continues until the end of the strad and then falls off. Two strands with primer one and primer two appear.


Base Pairs

A-T and G-C are base pairs. They are complementary nucleotides.

Base Pairs in Thermal Cycling

When the temperature dropped, the single strand DNA molecules to pair up but there are too many primer sequences than DNA strands. The primers overcrowd and lock onto their target before strands can rejoin. This causes the pairing of nucelotides. When the temperature increases, the DNA polymerase locates an attached primer with a single DNA strand, it begins to add a complementary nucleotide. It continues until the end of the strad and then falls off.

Name:PCR and explanation



SNP Information & Primer Design

Background: About the Disease SNP

SNP, also known as single nucleotide polymorphism, is when a base pair in a strand a DNA undergoes variation. The variation that was discovered in this lab was found on the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene which was found on the 6:131851228 chromosome. ENPP1 usual functions involve protein homodimerization activity, NADH pyrophosphatase activity, and nucleoside-triphosphate diphosphatase activity. A variation in this location results in the onset of Type 2 Diabetes in homo sapiens. The SNP of this disease changes alleles found at the position 131851228 which changes AAG to CAG which results in the disease.

Primer Design and Testing

The first primer test involved the non-disease forward primer, 5'-TTCAGATGACTGCAAGGACA-3', and the non-disease reverse primer, 5'-TGTTTAAAGTTTCTTTAAT-3'. When put into In-Silico PCR, the result was 220 bp. The forward primer's melting temperature was 57.9°C, and the reverse primer's melting temperature was 44.2°C. The second primer test involved the disease forward primer, 5'-TTCAGATGACTGCAAGGACC-3', and the non-disease forward primer, 5'-TGTTTAAAAGTTTCTTTAAT-3'. When put into In-Silico PCR, there were no matches for the corresponding primers. the disease forward primer had a melting temperature of 58.8°C and the reverse primer had a melting temperature of 44.2°C. Name: The Non-Disease Screen Capture

Name: The Disease Screen Capture

References

ENPP1 Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 [Homo Sapiens (Human)] - Gene - NCBI. https://www-ncbi-nlm-nih-gov.ezproxy1.lib.asu.edu/gene?cmd=Retrieve&dopt=Graphics&list_uids=5167#gene-ontology. Accessed 14 Mar. 2018.

How Jack Black Channeled His Inner Teenage Girl For Jumanji. 18 Aug. 2017, https://www.cinemablend.com/news/1693510/how-jack-black-channeled-his-inner-teenage-girl-for-jumanji.

Hsiao, Tun-Jen, and Eugene Lin. “The ENPP1 K121Q Polymorphism Is Associated with Type 2 Diabetes and Related Metabolic Phenotypes in a Taiwanese Population.” Molecular and Cellular Endocrinology, vol. 433, 15 2016, pp. 20–25, doi:10.1016/j.mce.2016.05.020.

Karen Gillan Speaks out after Fans Attack Her Skimpy Attire in Jumanji Reboot | Daily Mail Online. http://www.dailymail.co.uk/tvshowbiz/article-3801526/Karen-Gillan-speaks-fans-attack-skimpy-attire-Jumanji-reboot.html. Accessed 14 Mar. 2018.

Kasdan, Jake. Jumanji: Welcome to the Jungle. 2017, http://www.imdb.com/title/tt2283362/.

Neamati, Nahid, et al. “The ENPP1 K121Q Polymorphism Modulates Developing of Bone Disorders in Type 2 Diabetes: A Cross Sectional Study.” Gene, vol. 637, Dec. 2017, pp. 100–07, doi:10.1016/j.gene.2017.09.042.

“Polymerase Chain Reaction.” Wikipedia, 12 Mar. 2018, https://en.wikipedia.org/w/index.php?title=Polymerase_chain_reaction&oldid=830087355.

Reference SNP (RefSNP) Cluster Report: Rs1044498 ** With Other Allele **. https://www-ncbi-nlm-nih-gov.ezproxy1.lib.asu.edu/projects/SNP/snp_ref.cgi?rs=1044498. Accessed 14 Mar. 2018.

Rs1044498 - SNP - NCBI. https://www-ncbi-nlm-nih-gov.ezproxy1.lib.asu.edu/snp/?term=rs1044498. Accessed 14 Mar. 2018.

Severson, Tyler J., et al. “Genetic Factors That Affect Nonalcoholic Fatty Liver Disease: A Systematic Clinical Review.” World Journal of Gastroenterology, vol. 22, no. 29, Aug. 2016, pp. 6742–56, doi:10.3748/wjg.v22.i29.6742.

The Classic Robin Williams Movie Jumanji’s Kevin Hart Would Like To Work On Next. 15 Dec. 2017, https://www.cinemablend.com/news/1744820/the-classic-robin-williams-movie-jumanjis-kevin-hart-would-like-to-work-on-next.

UCSC In-Silico PCR. http://www.genome.ucsc.edu/cgi-bin/hgPcr?command=start. Accessed 14 Mar. 2018.