Polymerase Chain Reaction (PCR) is a method that use to make large quantities of a piece of Deoxyribose nucleic acid DNA artificially. The PCR technique has made it possible to analyze DNA fragments in samples that contain amounts of DNA that are either too small, or too degraded, to permit other types of nucleic acid analysis. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.
The PCR method is a cycling reaction in which template DNA is denatured by heating to separate the strands of the molecule. Primer (20-30 base fragment of DNA complementary to a region of the template) is annealed to the single-stranded templates. The cycle ends as the primer molecules are elongated by the action of DNA polymerase to produce molecules that are identical copies of the original template.
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PCR technology is unique in its ability to locate and exponentially amplify a small quantity of a specific nucleotide sequence which is "lost" against a large background of total nucleic acid. This feature of PCR has made possible the development of a vast number of experimental and diagnostic molecular biology techniques, which were previously extremely time consuming or, in many cases, impossible to perform.
In detail, PCR techniques consist of around 30 repeated cycles of temperature changes. Initialization step consist holding reaction of DNA piece, enzyme polymerase and primers at 94-96 Â°C temperature for 2-9 minutes. Further to this, denaturation of DNA strand takes place, in which DNA melts into single stranded DNA molecules. Next to this, annealing is done. In annealing step, temperature of reaction is being down to around 55 Â°C for 20-40 seconds. DNA-DNA hydrogen bonds are formed which are stable in nature. To the hybrid of primer-template, polymerase binds and results in synthesis of DNA. After this initiation, elongation of DNA is done. The temperature depends on the enzyme being used considering its thermal stability. New strand synthesis of DNA takes place which is complementary of the template. The direction of this extension is in 5' to 3' direction of DNA, thus 5'-phosphate group is condensed with 3'hydroxyl.
Materials and Methods:
ddH2O, Buffer, MgCl2, dNTP, PF1, PR1, Taq, Template DNA, micro-pippete, pipette tube, ethanol 70%, tissues, PCR microtube, PCR multiwall plates, agarose, gel rack, comb, TBE buffer 1x, Ethidium Bromide (ETBR), Load dye, 100bp marker, glove, microwave, balance, E-centrifuge, MS2 minishaker, Thermal cycler (PCR machine), AlphaImager.
Preparation of Mastermix
Master mix was prepared by mixing these reagents:
Reagen t Âµl
Template DNA 1.20
Master mix solution was centrifuged.
8.8Âµl of master mix was pippeted into each PCR microtube.
1.2Âµl of template DNA was pippeted into PCR microtube, using a new tip for each sample.
Microtube was mixed well. Spin for 5 seconds in microfuge to remove liquid from sides of tube.
Microtube was placed in PCR machine, program machine, and PCR reation was began.
Preparation of Agarose Gel
Agarose was weighted out 0.5g into a 250mL conical flask.
50mL of 1xTBE buffer was added and swirled to mix.
Agarose was dissolved for about 1 minute in a Microwave.
Agarose was leaved it to cool on the bench for 5 minutes down to about 60Â°C (just too hot to keep holding in bare hands).
1ÂµL of ethidium bromide was added and swirled to mix.
The gel was slowly poured into the tank. Any bubbles were pushed away to the side using a disposable tip. The comb was inserted and double check that it is correctly positioned.
Leaved to set for at least 30 minutes, preferably 1 hour, with the lid on if possible.
Staining the gel
2Âµl amount of load dye was added into each tube and the tip was leaved in the tube.
Then, the first well was loaded with 5Âµl of marker.
5Âµl of DNA was loaded into each tube.
The samples were continued loaded and finished it with a final lane of marker.
Always on Time
Marked to Standard
Used TBE poured into gel tank and gel was placed.
The gel tank was closed, the power source was switched on and the gel was ran for 30 minutes (85V, 250Wma, 100Watt).
The progress of the gel was monitored by refererence to the marker dye.
Switched off and unplug the gel tank and observed the gel under AlphaImager.
Band PCR 16s
The experiment was done on 19 samples of Pangasius sp. From the Table 1, only 5 samples of Pangasius sp. showed the band. There were PH 64 (Patin buah), PH 7 (Patin juara), PH 20 (Patin buah), PH 23 (Patin buah), and also PH 52 (Patin juara). So, the samples were suitable as a candidate for next stage which is sequencing. The others samples of Pangasius sp. showed no band. It is because maybe the primer that used (2F and 2R) is not suitable for the other 14 samples. The others primer such as 162F and 162R maybe can be used for the others samples. PCR program that were use for this experiment was 16sAyong. There are 6 process in PCR program which are initial denaturation, denaturation, annealing, extension. Final extension, and also hold process. Initial denaturation takes 3 minutes (94Â°C), denaturation takes 0.25 minutes (94Â°C), and annealing takes 1.0 minutes (50Â°C). After that, extension process was done for 1.0 minutes (72Â°C), and then was followed by final extension for 7 minutes (72Â°C). Hold process were the final step that happen in 40Â°C (âˆž).
As a conclusion, specific PCR band for Pangasius sp. using 16sRNA were indentified for 5 species.
Thermal cycler Micropippette Alpha Imager
Ethidium Bromide Centrifuge 1x TBE Buffer