BME100 f2014:Group25 L4

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OUR GROUP

Angus Cheung
Brandon Dorr
Ben Drotar
Daniel Gentry
Srekar Nagishetty Ravi
Diana Tran

BME100: PCR LAB A

Materials

  • Disposable Gloves
  • Laboratory Coat
  • PCR Reaction Mix, 8 Tubes, 50 microliters each
  • DNA/primer Mix, 8 Tubes, 50 Microliters each
  • Strip of Empty PCR Tubes
  • Disposable Pipette Tips
  • Container for used Pipette Tips
  • Micropipette
  • OpenPCR machine

Note:

  • PCR Reaction Mix is composed of Taq DNA polymerase, Magnesium Chloride, and dNTP's.
  • DNA/primer mix is composed of a DNA template (each group has a different template), along with a forward and reverse primer.
  • Do NOT use Disposable Pippette Tips more than once.

PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G25 + Positive control none
G25 - Negative control none
G25 1-1 Patient 1, replicate 1 21611
G25 1-2 Patient 1, replicate 2 21611
G25 1-3 Patient 1, replicate 3 21611
G25 2-1 Patient 2, replicate 1 21095
G25 2-2 Patient 2, replicate 2 21095
G25 2-3 Patient 2, replicate 3 21095

DNA Sample Set-Up Procedure

  1. Gather your materials, making sure not to open either of your controls.
  2. Cut the 8 PCR tubes in half.
  3. Label the tubes based on the DNA they contain as well as the primer they contain.
  4. Fill each of the tubes with 50 µl of the PCR mix.
  5. Add 50 µl of DNA (making sure to add the correct DNA to each tube based on its label) to each of the 8 tubes. Make sure to use a new pipette tip for each sample of DNA. There should now be 100 µl of solution in each of the tubes.
  6. Place your PCR tubes in the thermal cycler. Before starting the machine make sure that the PCR thermal cycler is completely full. This requires two groups worth of tubes.
  7. Use the OpenPCR Program to start the PCR thermal cycler to 35 cycles.

OpenPCR Program

Heated lid: 100°C
Initial step: 95°C for 2 minutes
Number of cycles: 35
-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

Question 1:

The template DNA is a DNA strain that holds the goal DNA region to be copied. The primers are nucleic acids which mark the starting ends of replication. Taq polymerase is an enzyme which matches nucleotides and creates a single strand of DNA. Deoxyribonucleotides (dNTP’s) are the building blocks of DNA that are put together by the polymerase enzyme. These include adenine, thymine, guanine and cytosine.


Question 2:

In PCR, DNA is first heated to 95°C for 3 minutes. During this time Taq polymerase gains energy and becomes activated and DNA strands become stressed and begin to denature. Next, the temperature stays at 95°C for 30 seconds at which time double stranded DNA denatures into two single strands. After this, the solution is cooled to 57°C for 30 seconds which causes annealing, where with proper base-pairing, primers bind to the matching segments on the single stranded DNA target segments. After this, the solution is heated to 72°C for 30 seconds so that the DNA can be extended by taw polymerase which binds to the primers and adds nucleotides to copy the DNA by properly base-pairing the nucleotides to the single stranded DNA. This is completed a total of 35 times before bend held at a constant 72°C for 3 minutes which allows for any remaining single stranded DNA remnants to be completed with a continued extend phase. Lastly, the solution is cooled to 4°C in order to improve the storage of the DNA.

Question 3:

Which nucleotide bases bind to each other?

  • Adenine (A): T
  • Thymine (T): A
  • Cytosine (C): G
  • Guanine (G): C

Question 4:

When do nucleotide bases pair?

During the Anneal and Extend phases of thermal cycling base-pairing occurs. Base-pairing between the DNA and primers occurs during the Anneal phase and pairing between nucleotides and the DNA assisted by the Taq polymerase.

Bonus

Bonus.jpg

BME100: PCR LAB B

20-200 μL Micropipettor Challenge

Tube Red Blue Green Yellow Final Color Final Volume (μL)
1 30 μL 20 μL 0 μL 0 μL Violet/Dark Purple 50 μL
2 20 μL 0 μL 0 μL 30 μL Orange 50 μL
3 0 μL 50 μL 0 μL 50 μL Green 100 μL
4 0 μL 0 μL 50 μL 50 μL Green 100 μL

Figure 1
Questions
1. Which of the data is quantitative and which is qualitative? (*Note: Qualitative data = descriptions; Quantitative Data = Specific Measurements)
* The quantitative data includes the volumes of each color needed as well as the final volume since these values are explicitly numerical and serve to represent and measure volume (e.g. 5 mL, 10nM, 50mM, 100 μL, etc.). In this case, we are using μL (microliters) as our unit of measurement.
* The color of the food coloring suspensions and the final color of the combined solutions are examples of qualitative data because they describe the state and condition of the sample (e.g. blue, rough, shiny, bubbly, etc.). Specifically, we are using qualitative color descriptors.
2. What is the difference between accuracy and precision? Let's take a look at a dart board.
Dartboard.jpg
Figure 2.Image from: [1]

  • The goal is to hit the dartboard in the very center, at the bullseye. If you hit the dartboard consistently in one general area, you are accurate. The capability of reproducing the same data shows accuracy. If you hit the dartboard at the bullseye, you are precise. The capability of obtaining the desired value/data set illustrates precision, marked by exactness. If you hit the dartboard at the bullseye every time, you are accurate precise -- showing consistency and desirability. Refer to Figure 3 below.

AccuracyPrecisionDartBoard.jpg
Figure 3. Image from: [2]
3. In this experiment, were you accurate, precise, or both?

  • In this experiment, we were precise. Since we did not run the trial multiple times, we cannot determine our accuracy. However, we were precise because our micropipettors measured exact measurements multiple times in order for us to create the food coloring suspensions.

Lab Scenario B

Several patients have submitted DNA to be tested for a disease marker.
*DO NOT FORGET TO LABEL YOUR SAMPLES APPROPRIATELY. IF YOU MIX UP OR MISLABEL YOUR SAMPLES, YOU COULD MISDIAGNOSE A PATIENT.

Goal

Run Polymerase Chain Reaction (PCR) reactions on DNA samples.

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix (8 tubes, 50 μL each; Mix contains Taq DNA-Polymerase, MbCl2, and dNTP's)
  • DNA/Primer mix (8 tubes, 50 μL each; Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer.)
  • A strip of empty PCR tubes
  • Disposable pipette tips.
    *Only use each only once. Never re-use disposable pipette tips or samples will be cross contaminated*
  • Cup for discarded tips
  • Micropipettor
  • Open PCR machine
    *Each machine will be shared by two groups*

Initial Machine Testing

The Original Design


Experimenting With the Connections


Test Run



Protocols

Thermal Cycler Program


DNA Sample Set-up

row 1 cell 1 row 1 cell 2 row 1 cell 3 row 1 cell 4
row 2 cell 1 row 2 cell 2 row 2 cell 3 row 2 cell 4


DNA Sample Set-up Procedure

  1. Gather all materials.
  2. Cut the strip of empty PCR tubes in half. There will be two strips of four linked tubes. This is necessary to fit all of your tubes in the OpenPCR machine.
  3. Use a black marker to label the sides of the empty tubes with the Tube Labels your group created. * Do not label the lids of the tubes
  4. Place the tubes in the rack.
  5. Start with the empty tube you labeled as the positive control. Using proper pipetting technique, transfer 50 μL of PCR Reaction Mix into this empty tube. Discard the disposable tip into the collection cup. Do not re-use tips and cross-contaminate your samples!
  6. Using a fresh pipette tip, transfer the positive control DNA/primer mix into the same tube. The total volume in your positive control PCR reaction tube is now 100 μL.
  7. Repeat steps 5 and 6 for the negative control. Use the appropriate DNA/primer mix for the corresponding tubes. When you are done, all of your labeled tubes should contain DNA/primer mix, resulting in a 100 μL complete PCR reaction in each tube.
  8. Close the lids tightly on the PCR reaction tubes.
  9. Take the tubes over to your assigned PCR machine. Place the tubes into the slots in the heating block. Do not start the machine until all 16 slots are filled (multiple groups need to run the reactions simultaneously).


PCR Reaction Mix

  • Taq DNA-Polymerase
  • MgCl2
  • dNTP's


DNA/ primer Mix

  • Template DNA (Each sample has a different sample of template DNA)
  • Forward primer
  • Reverse Primer



Research and Development

PCR - The Underlying Technology





BME100: PCR LAB D

What is a Nucleotide?

  • Nucleotides form nucleic acids such as DNA and RNA. They serve as subunits to nucleic acids. There are four types of nucleotides: Adenine, Guanine, Thymine, Cytosine, and Uracil. They have at least one phosphate group and thus are able to move energy within the cell in the form of nucleoside triphosphates.


What is a polymorphism?

  • Polymorphism is the genetic variation in the DNA within a population. The genetic variation is caused by mutations in the nucleotides. Natural selection relies on polymorphism in order to be able to bring substantial change to a population.


What species is this variation found in? (latin name)

  • Homo sapiens


What chromosome is this variation found on?

  • 21:34370656


What is listed as the Clinical Significance of this SNP?

  • Pathogenic


What gene(s) is this SNP associated with?

  • KCNE2


Click the PubMed link to view summaries of research associated wit the SNP. What disease is linked to this SNP?

  • Congenital long QT syndrome


What does KCNE2 stand for?

  • Potassium-voltage-gated channel, lsk-related family, member 2


Click the KCNE2 link. Briefly describe the molecular functions of this gene?

  • The gene regulates neurotransmitter release, heart rate, insulin secretion, neuronal escitability, epithellal electolyte transport, smooth muscle contraction, and cell volume. The gene encodes a member of the potassium channel, voltage-gated, lsk-related subfamily.


What is an allele?

  • An alternate form of a gene which arises by genetic variation. It is any of the possible forms in which a gene for a specific trait can occur. Generally one allele is inherited from each parent. If the paired alleles are the same then its called homogenous, and if they are different then they are called heterozygous. Most alternate forms of the gene do not show any observable changes in the phenotype.


The disease-associated allele contains what sequence?

  • CTC


The numerical position of the SNP is?

  • Position 34370656


Non-disease forward primer (20 nt)

  • CAT GGT GAT GAT TGG AAT GT


The numerical position exactly 200 bases to the right of the disease SNP is

  • Position 34370856


Non-disease reverse primer (20 nt)

  • CCC TTA TCA GGG GGA CAT TT


Disease forward primer (20 nt)

  • CAT GGT GAT GAT TGG AAT GC


Disease reverse primer (20 nt)

  • CCC TTA TCA GGG GGA CAT TT