Amplification of DNA by the Polymerase Chain Reaction & DNA Electrophoresis

Abstract:

In the polymerase chain response, it was consolidated with gel electrophoresis to find the “Alu gene” in DNA using transposons. Polymerase Chain Reaction utilizes certain elements to imitate certain duplicates of a DNA arrangement to help give the gel more “Alu squences” to be resolved simpler. Transposons take into consideration the most widely recognized Alu quality to be perceived using the gel electrophoresis. Each human might possibly contain a chromosome containing the alu quality and some may or may not even have one chromosome that contains the genome. The Polymerase Chain Reaction combined with gel electrophoresis takes into consideration the specific gene that will be identified and in our personal samples to be either homozygous/heterozygous or negative/ positive. 

Introduction:

“Polymerase chain response”, also known as PCR, utilizes DNA polymerase to reproduce another strand of DNA from a supplement format. The response needs the accompanying response blend parts: Deoxynucleotides (ddNTPs), Chromosomal DNA, and Taq DNA polymerase; and additionally reverse and forward primers. The thermocycler was required to begin the procedure through the means of tempering, expansion and denaturing. Denaturing utilizes warmth to isolate DNA strands, toughening places the forward and switch preliminaries to set up the expansion stage and the taq DNA polymerase prolongs the strands of the DNA in the expanded stage.

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The bits of DNA are “transposons” that can supplant itself in somewhere else in the genome. It is where it can recreate and basically reorder itself into another DNA. “Double-stranded DNA reinsert elsewhere in the genome, i.e., the classic “cut-and-paste” transposons” (Cédric) The accessible and the most available transposons are Alu components which are in the 300bp range. ALu genes are a standout amongst the most copious found in the “human genome at roughly 1 million” (Prayel).

The technique in which DNA strands are assessed by length is obtained by using “Gel Electrophoresis”. Every model is put into a well in the agrose gel where it is subjected to electrical current to ultimately have the DNA strands move towards the anode or positive charge bearing. “Shorter strands can push ahead on account of the manner in which that the gel contains sugar fibers that can obfuscate longer strands and its movements” (Heuer).

As every human is different, some may or may not have the alu gene. Some may even be carriers of the alu gene (Hetrozygous), maybe they have obtained the alu gene from only one of their parents and not the other. Whereas other that are homologous negative, have not received the alu gene from either one of their parents.

Methods:

Everyone in the lab will take an example of their cheek cell and break it down through using the PCR method by utilizing a toothpick and scratching within the sides of the cheek/mouth. Once done, put the finish toothpick that you scratched your cheek cells with. Use a microcentrifuge to obtain .3 ml and mix it with 100 uL of the buffer extraction and whirl it around. Name and top the test tube to exchange to the warm cycler at fifty-five degrees celsius for an hour. Towards the finish of the cycle, for approximately ten minutes, it will be at ninty-five degrees Celsius to inactivate the protein. Recoup and ensure the test tube is legitimately named.

Start to set up the PCR by marking a tube “alu” with your initials and add .2 microliters. Next, add the following: 7.5 uL of distilled water, 1.25 uL of forward primer, 1.25 uL of the reverse primer, 2.5 uL of all the cheek cell genomic DNA, and 12.5 uL of the “Master Mix”. Each table will set up a control .2 microliters of the PCR tube and name it “C” with their initials and instead of adding the genomic cheek cell DNA will be adding 2.5 uL of distilled water. Set the examples in the thermocycler and sit patiently for the teacher to begin it for thirty-five cycles. Record the temperatures and times through one finish cycle 3 times. The denaturing step was set at ninty-five degrees Celsius for an entire hour, and for 60 seconds at sixty-two degrees Celsuis was the annealing step and the seventy degrees Celsius for 60 seconds was also for the extending step.

Proceed forward and obtain the sample, utilizing the class size, make sure the class samples can fit the considerably even # of gels. With the fourteen-well comb put 1.5 percent of agarose gel and give it time to solidify totally. Setting the gels in the electrophoresis chamber closest the dark anodes put 1X TBE and expel the brush. In the most remote left and right segments embed bp ladder of 6 ul. Now, in everyone’s 20 microliter sample, add “four uL” of the dye and from the mixture put “five uL” in the gel. Run the gel for half-an-hour at 120 volts. Once completed, take the gel to the region specified by your TA to put the gel in an UV light box and to see the photo of what is shown.

Results:

The outcomes of this lab demonstrated that various models had particular results in that some were either negative or positive for the gene of the alu. And some could actually be carriers of the gene, and be “heterozygous”.  Only four students that were on the left side will be are area of interest for this result section.

As for the markers that were outside wells, “seven and one”, the well four was the control. And well “one and three” showed the alu gene negative homozygous meanwhile for well 4, there was a “alu gene” that was positive homozygous. Also, nothing was showed on the gel for well two and well 3. None of the models were seen to be heterozygous subject to the undeniable social occasions that could be surely watched.

By utilizing the gel electrophoresis, the photo shows the distinctive alu gene pieces. Wells “two through six” were one gathering of four understudies that will be inspected further and wells “nine through twelve” was another gathering of three understudies. Wells “one, seven, eight and thirteen” contained every single marker while “four and ten” were the controls of the examination. Sadly, the personal sample had no alu gene shown on the gel. Wells “two and four” contained groups at the 400bp implying that the examples were negative homozygous for the gene of the alu in the two chromosomes. Well “six” contained another example in which was positive homozygous for the gene of the alu in the two chromosomes.

Discussion:

As for the “Polymerase Chain Reaction”, the results can be better furthered and can be bettered improved through the utilization of the recently created “microfluidic systems”, in which multiple, various reactions can occur simultaneously at once. The Real-time “Polymerase Chain Reaction”, PCR are can be finished in about an hour or less, or, in other words, way faster than the ordinary PCR that has been utilized in this lab. Through the utilization of the “real-time PCR”, it allows for a quicker diagnosis of infectious disease that would otherwise be fatal if it is not figured out in a timely manner (Ahrberg).

The flaws that could have occurred for this lab are infinite and could have occurred at any given time during the duration of the experiment. For instance, the measurements could have been off and as a result, skew the whole outcome of the experiment. Or a simple mistake in the beginning of the lab, not taking enough cheek cells on the toothpick. This may have been because there was a lack of scraping correctly on the sides of the insides of the mouth. As a result, the lack of cells on the toothpick couldn’t be used for the PCR to see the alu genes.

As for the sample that was personal, the photo shows nothing on wells three. Wells two and five had contained samples that showed homogenous negative for the gene of the alu. Meanwhile, for the well six, it showed that the sample was indeed positive homogenous for the gene of the alu. The bands that were faint were not shown because it was impossible to legitimately have the capacity to tell which tests were homozygous or heterozygous for the gene of the alu for those that were faint in color.

Conclusion:

The hypothesis was not ready to be tried in light of the fact that the individual sample was not found or done inaccurately. In any case, utilizing the first sample one, the outcomes were valid that the example would be a homozygous negative for the gene of the alu. The identification of the gene depended entirely upon the individual samples on whether or not each sample had a chromosome for the alu gene. “Polymerase Chain Reaction”, PCR is imperative since it recreates DNA strands using distinctive parts. PCR is precise in separating the DNA strand than reconstructing another supplement.

References:

• Feschotte, Cédric, and Ellen J. Pritham. “DNA Transposons and the Evolution of Eukaryotic Genomes.” Current Neurology and Neuroscience Reports., U.S. National Library of Medicine, 2007,
• Ahrberg, C D, et al. “Polymerase Chain Reaction in Microfluidic Devices.” Current Neurology and Neuroscience Reports., U.S. National Library of Medicine, 5 Oct. 2016
• Alberts, Bruce. Essential Cell Biology. New York, NY: Garland Science Pub, 2017. Textbook.
• Prayel, Leslie A. “Functions and Utility of Alu Genes.” Nature News, Nature Publishing Group, 201
• Heuer, Tim. “PROTEIN GEL ELECTROPHORESIS.” Oligo-DT Cellulose Columns, 1998, 

Questions:

SECTION B:  PCR Amplification of Alu Sequences

Q1) Proteins are denatured at high temperatures. So how are we able to perform PCR at these high temperatures using DNA copying enzymes?

We were able to do this because it had a thermostable enzyme called Taq polymerase, in which could not denature at high temperatures.

Q2) What was the annealing temperature that was programmed into our thermocycler? Why was this temperature chosen?

The temperature was sixty two degrees celsius, and the reasoning behind that was because the primers had to start synthesizing DNA at the low temperatures.

Q3) If the sequences of the PCR primers are;

5’-GGA TCT CAG GGT GGG TGG CAA TGC T-3’

5’-GAA AGG CAA GCT ACC AGA AGC CCC AA-3’

Calculate the melting temperature of each, based on the observation that for every A or T add 2^oC, and for each C or G add 4^oC?   Show your calculations.

For the 1st strand we had to do:

–          Guanine/Cytosine=15

–          Adenine/Thymine=10

–          15×4 +10×2=80 C

For the 2nd Strand we had to do:

-Guanine/Cytosine=14

– Adenine/Thymine=12

-14×4 +12×2=80 C

Q4) If the extension temperature was eliminated altogether from the thermocycler, what would happened to our Alu PCRs? [Hint: the size of your fragments is important]

The alu PCRs will would become shorter because during the extensions stage, the DNA will become extended so that they don’t shrink. This is to ensure that the final product once done will display the alu genes, showing whether it’s is a either a negative/ positive for the heterozygous or homozygous.

 SECTION C:  Electrophoresis of Amplified DNA

Comprehension Questions:

Q1. What is a transposon?

A transposon, in essence, is a piece of “Deoxyribonucleic Acid”, DNA that that can replace itself in the DNA sequence.

Q2. How common are transposons in the human genome?

Interesting to note, Transposons are common in the human as there are approximately over a million of them in your very genome.

Q3. How does a transposon “jump” from one site in the genome to another?

They jump from site to site in the genome by a method that is called, “copy and paste”. This helps replicate itself using the mRNA and then cut out DNA pieces and incorporates itself inside.

Q4. What is a polymorphism?

Polymorphism is variation in the DNA’s sequence in which is a common in the population.

Q5. How can PCR be used to determine the Alu genotype of an individual?

PCR is utilized to determine the alu genoype of an individual by the use of gel electrophoresis. By using gel electrophoresis, the alu genotype of a person can be seen in the band length and therefore show whether a sample is positive or negative for the alu gene.

Q6. How is Taq DNA polymerase different from DNA polymerase found in E. coli?

Taq DNA polymerase is different from DNA polymerase found in E. Coli in that , the Taq is a lot quicker in the breakdown of the DNA by using high temperature.

Q7. What is the role of the forward and reverse primers used in PCR?

One of the main roles of the “forward” and “reverse” primers allows the DNA’s target reason to replicate.