In a Polymerase Chain Reaction (PCR), a specific sequence of DNA is amplified and multiplied. PCR requires Taq DNA Polymerase, deoxyribonucleotides, template DNA, primers, and a reaction buffer. In this lab we analyze PCR reactions to test two patients for the presence of a known pathogen. The process begins with the mixing of the template DNA, Taq Polymerase, and primers into a tube. The tubes are then sent to a thermocycler to be heated to separate the DNA, from there the primers for the target DNA bind and amplify the segments of the targeted DNA. This makes it possible to detect the presence of the pathogens in the patient DNA samples.
During the setup of our reaction, the pre-lab was very informative and helpful in explaining how to complete the micropipetting for both patients and their replicate DNA. We had trouble differentiating between the first and second stop, though it only took a few tries to get the hang of it, and then it was easy to complete the rest of the setup. We were careful in labeling so that we did not have to go back to our first write-up and change the labeling scheme. There were no errors in labeling, and the setup went well overall. The final reactions had the same amount of liquid, and there was a very small amount of liquid left in the tubes that held the DNA samples and PCR reaction mix.
Fluorimeter Procedure
Imaging set-up
The following procedures were completed during the PCR experiment. First, a fluorimeter kit was obtained, including: black box, fluorimeter, phone stand, glass slides. Then, two green micro test tube containers were placed on top of each other in order to get the fluorimeter to line up with the phone camera. The glass slide was placed inside the fluorimeter from the back with the glass slide facing down and the rough. hydrophobic side with the dots going towards the front of the fluorimeter. The camera used was an iPhone 6, with the flash settings turned off. The iPhone was held up using the phone stand that came with the fluorimeter kit. The black box was used to create a dark environment for the iPhone imaging.
Placing Samples onto the Fluorimeter
Set pipette to 80 microliters
Pipette 80 microliters of the desired DNA sample (water, 0.25, 0.5, 1.0, 2.0, 5.0) and then drop it onto the middle of the glass slide
Remove pipette tip and get a new tip
Pipette 80 microliters of SYBR Green 1 solution and then add it to the drop on the middle of the glass slide
Move the black box over the fluorimeter set-up to create a dark environment, then use the iPhone to take three images of the drop, with the drop in focus.
After three images of the drop are taken, all the liquid is pipetted off and then disposed of in a waste beaker.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
ImageJ: Zero DNA
ImageJ: 0.5 μg/mL sample (low calf thymus DNA)
ImageJ: 5.0 μg/mL sample (high calf thymus DNA)
Calibrator Mean Values
Table 1: Calibrator Raw Data
Sample Number
Image Number
Final DNA concentration in SYBR Green 1 solution (µg/mL)
Area
Mean Pixel Value
RAWINTDEN of the drop
RAWINTDEN of the background
Calibration 0.0
1
2.5
47956
86.441
4145365
200938
2
2.5
47956
82.417
3952380
202200
3
2.5
47956
82.699
3965899
213770
Calibration 0.25
1
1
47956
113.946
5464414
209035
2
1
47956
113.569
5446291
208512
3
1
47956
115.439
5536011
204692
Calibration 0.5
1
0.5
47956
149.951
7191061
213014
2
0.5
47956
149.709
7179447
208211
3
0.5
47956
152.512
7313874
213955
Calibration 1.0
1
0.25
47956
214.836
10302664
208531
2
0.25
47956
210.562
10097702
201800
3
0.25
47956
216.349
10375213
201307
Calibration 2.0
1
0.125
47956
238.391
11432268
208362
2
0.125
47956
234.668
11253742
229573
3
0.125
47956
234.405
11241115
226871
Calibration 5.0
1
0
47956
238.327
11429210
213803
2
0
47956
238.483
11436712
212428
3
0
47956
237.795
11403679
210607
Calibration curves
Images of Our PCR Negative and Positive Controls
ImageJ: Positive Control
ImageJ: Negative Control
PCR Results: PCR concentrations solved
Table 5: PCR Solved
PCR Product Tube Label
Mean of RAWINTDEN Drop - Background
PCR Product Cocentration (µg/mL)
Total Dillution
Initial PCR Product Concentration (µg/mL)
Positive Control
10980608
72240833.16
12
866889997.9
Negative Control
4281269.667
28166238.86
12
337994866.3
Patient 1 - Sample 1
4395681.333
28918947.19
12
347027366.3
Patient 1 - Sample 2
6176889.333
40637420.88
12
487649050.5
Patient 1 - Sample 3
2594612.333
17069809.04
12
204837708.4
Patient 2 - Sample 1
11699727
76971879.21
12
923662550.5
Patient 2 - Sample 2
10974659.67
72201699.39
12
866420392.6
Patient 2 - Sample 3
11208122.67
73737640.18
12
884851682.1
PCR Results: Summary
Our positive control PCR result was 866889997.9 μg/mL
Our negative control PCR result was 337994866.3 μg/mL
Observed results
Patient 54647: These samples when qualitatively observed, look similar to the negative control sample results because there was very little green in the drops when observed in the dark. When quantitatively observed, the sample results for this patient are also very similar to the negative control sample because the initial PCR Product Concentration for the negative control is 337994866.3 μg/mL and the average initial PCR Product Concentration for this patient is 346504708.4 μg/mL which are very similar values.
Patient 67052: These samples when qualitatively observed, look similar to the positive control sample results because there was a lot of green in the drops when observed in the dark. When quantitatively observed, the sample results for this patient are also very similar to the positive control sample.When quantitatively observed, the sample results for this patient are also very similar to the positive control sample because the initial PCR Product Concentration for the positive control is 866889997.9 μg/mL and the average initial PCR Product Concentration for this patient is 891644875.067 μg/mL which are very similar values.
Conclusions
Patient 54647: Negative; This patient does not have the disease because their samples show little to no evidence of having the DNA sequence associated with this disease.
Patient 67052: Positive; This patient does have the disease because their samples show evidence of having the DNA sequence associated with this disease.
BME 100 Fall 2017
