Finding Pork DNA

Question:
What is the process that can find pork DNA?

Variable
Independent Variables: the type of DNA (pork DNA and chicken DNA)
Dependent variables: only the color regions of pork DNA will appear on the gel after Agarose gel electrophoresis
Controlled variables: Method (ex. temperature, concentration of condition and neatness during the process), time during Agarose gel
electrophoresis

Hypothesis:
If we want to find the pork DNA, then we can use three process that are distilling DNA, PCR, and Agarose gel electrophoresis.

Background Research
DNA (Deoxyribonucleic acid) is the hereditary material in almost organisms. Most DNA are found in cell nucleus and some in mitochondria.
DNAs structures are formed by two long strands of polynucleotides bind
in spiral called double helix. The genetic code of organism depends on the base sequences on the two strands of DNA. Some chemical, high
temperature or X-ray can break down the bonding of DNA strands. PCR (polymerase chain reaction) is a process of a particular DNA synthesis
in the experimental tube by the imitation of the natural DNA synthesized in organism. These techniques are developed by Karry Mullis. Most of
researchers used these techniques to generate more amount of hereditary materials. There are 3 principal steps in PCR; First step is denaturing,
a step of separating DNA which has the double strands by gradually increasing the temperature until it becomes a single strand DNA, by keeping
the temperature between 90-95 degree Celsius. Second step is annealing, a step to decrease the temperature gradually and put the primer
(short DNA) into the system in order to bind the complementary base pair of primer and template DNA, by keeping the temperature between 37-
60 degree Celsius. Third step is extending, a step that has to put the DNA polymerase into the system in order to synthesis a new strand DNA or
increase the amount of DNA, by keeping the temperature between 72-75 degree Celsius. We can separate the type of DNA because each DNA
of pork and chicken will have the different size even though agarose gel electrophoresis is test that can separate the size of DNA. Due to inside
the gel, have many holes and the size of the holes depends on the ingredient of the gel that we can determine it. If the DNA is very tiny, DNA will
spread out of the hole but if DNA is very large, it cant pass into the hole. So, DNA has to be in the same size with the hole.
Materials (first process)
- Chicken
- Pork
- Microtubes (like a test tube)
- grinder (use to grind the meat)
- Micropipette
- Incubation box (to change temperature)
- Centrifuge (a spinning machine)
- Nanodrop (to check the amount of DNA) Chemicals used in the experiment (the first process)
- lysis buffer
- SDW (water)
- proteinase K
- phenol/chloroform/genezol
- isopropanol
- 70% ethanol

Distilling DNA
1. Put the pork and chicken, mix distilled water (400 L), and put them in the grinder to grind the pork and chicken
2. Use micropipette to absorb the liquid from mixture in 1 to the micro tube (200 L). After that, add lysis buffer(400 L) and proteinase K (2 L)
3. Put them into the incubation box at 65 C for 15 minute
4. Add phenol/chloroform
5. Put enezol (600 L)
6. Put them into the centrifuge to spin 12,000 times for 10 minutes.
7. Then, take upper phase into the new tube. Then, add isopropanol (400 L)
8. Then, put them into the centrifuge to spin 12,000 times for 10 minutes.
9. Discard supernate and add 70% ethanol (500 )
10. Then, put the tubes in the centrifuge to spin 12,000 times for 10 minutes.
11. Pour the liquid out and clean the tube with tissue
12. Incubate the DNA pellet at 37 C for 15 minutes
13. Then, dissolve DNA with SDW
14. Measure DNA quantity by Nanodrop.
Materials (the second process)
10 uM F
10 uM R
10x PCR buffer
10 mM dNTP
SDW
Taq DNA polymerase
DNA
Pipette
PCR machine

PCR(increase the amount of DNA)

1. Add 10 uM F (1 L)
10 uM R (1 L)
10x PCR buffer (2.5 L)
10 mM dNTP (1 L)
Taq DNA polymerase (0.5 L)
DNA (2 L)
SDW (17 L)
A total of 25 L in the different tubes with different types of DNA (pork and chicken)
2. Put them into the PCR machine for 2 hours

Materials (the third process)

- Agarose gel powder
- Buffer
- Tray
- Six plastic sticks
- Glycerol
- Food colors
- Pipette
- Water
- Ethidium bromide
- Ultra Violet light
Agarose gel electrophoresis
1. Prepare the gel by mixing agarose gel powder (0.45g.) and buffer(100g.) and dissolve them
*we put buffer, not water, because buffer can help to spread electric currents
2. Pour the gel on the tray that has comb. Then, we got six wells (tiny holes)
3. Drop the Glycerol and food color in small drop. Then, use a pipette to mix DNA, Glycerol, and colors together. It will become DNA sample.
*If we do not mix DNA with Glycerol, it will drift from the holes to buffer. Moreover, we add food colors to the DNA because it will help us to see
the DNA easier.
4. Put the DNA marker (the base sequence that show identity of each species)
5. Put 30 L of the pork DNA and chicken DNA that are mixed with Glycerol and food color into the different holes
6. Start to run gel for 50 minutes by using electric current 100 volt (the process will spread electric currents into gel to transfer DNA)
7. Put the gel tray into ethidium bromide for 5 minutes (it will bind into the DNA strand)
8. Put it into water to dissolve ethidium bromine for 10 minutes
*because if we do not dissolve it then it will get ethidium bromide contaminated with the gel.
9. After that, put the gel on the ultraviolet light

Future directions
After we know the DNA of pork and chicken, we can test Halal food (Muslim food) by finding pork DNA. But the pork DNA that we have
research is raw meat. So the pork that have been cooked with the food, the pork DNA will be an incomplete strand therefore we cannot test the
pork DNA in Halal food or food. Therefore, we must find a way to test the cooked pork DNA in the food or Halal food in order to know whether
there is pork in the food. According to JGU (Johannes Gutenberg Universitt Mainz), Almost all foodstuffs contain the genetic material of those
animal and plant species that were used in their preparation. Scientists at the Institute of Molecular Genetics, Genetic Security Research and
Consulting at Johannes Gutenberg University Mainz (JGU) have developed a novel screening procedure that provides for highly sensitive,
quantifiable analysis of animal, plant, and microbial substances present in foodstuffs. For this, the researchers have adapted the latest
techniques of DNA sequencing, which are otherwise currently employed in human genetics to unravel the genetic information of thousands of
experiments. Because of its potential, the method dubbed 'All-Food-Seq' by its developers has already attracted the attention of food
inspection experts. "This method is very interesting in connection with efforts to promote the molecular traceability of food," said Hermann Broll of
the German Federal Institute for Risk Assessment in Berlin and Dr. Ren Kppel of the Zurich Cantonal Laboratory in Switzerland. The method
developed by the Mainz scientists is thus to be validated in comparison with conventional detection techniques in the near future.
Conclusion
When the gel was put on the ultraviolet light, the orange lines that were pork DNA appear, but the chicken DNA did not appear. This is
because the gel that we used was the pork tasting DNA. During the experiment, we had learned a lot of the new things, such as how to find the
DNA. This process was divided into 3 parts: distilling DNA, PCR, and running gel. However, this experiment also had an error l because one of
the regions of chicken DNA appeared. In the reality, only the color regions of pork DNA should occur, but one of the color regions of chicken
DNA occurred. Therefore, if we have a chance to do the experiment again, we will be careful about using instruments because they may have an
accident that will affect our experiment result.

Acknowledgement
We are grateful for advice from Assist.Prof.Dr. Panan Kanchanaphum and Suthep Mongkollertlop at Rangsit University.
This work is supported by Suthep Mongkollertlop, the Science Medical Institute Biological Education in Rangsit University

References

Hankeln, T. (2013). New DNA test identifies ingredients in foods. Retrieved at 14th May 2016 from http://www.uni-
mainz.de/presse/16278_ENG_HTML.php

Promega (n.d.). Methods for Extracting and Identifying DNA from Food. Retrieved at 14th May 2016 from
https://worldwide.promega.com/resources/pubhub/tpub_130-analyzing-dna-in-food-samples/

ThaiBiotech.info (2009 - 2016). What is DNA. Retrieved at 13th May 2016 from http://www.thaibiotech.info/what-is-dna.php

ThaiBiotech.info (2009 - 2013). What is PCR. Retrieved at 13th May 2016 from http://www.thaibiotech.info/what-is-pcr.php