Gel Electrophoresis

DNA samples obtained can be separated according to the size of the fragments by gel electrophoresis.Gel electrophoresis is one of the most useful means of separating and purifying DNA fragments for further analysis. DNA samples extracted from any specimen can be amplified using PCR and then subjected to gel electrophoresis. The DNA sample is treated with a restriction enzyme to break it down to fragments. Following gel electrophoresis, all the sizes of the fragments of the given DNA can be collected and sequenced using a DNA Sequencer to reveal the genetic make-up of the given specimen. The following image and video illustrate the principle of gel electrophoresis.

            

Gel electrophoresis is also applicable for sorting RNA and proteins by size. The laboratory procedure for carrying out DNA gel electrophoresis is seen below.

            

Total DNA Extraction Protocol

Procedure:

1. Clean specimen and obtain a small sample for DNA extraction and place it in a sterile centrifuge tube.

2. Add 600μl nucleolysis solution to the sample and grind using a motorized pestle till the tissue degenerates. 

3. Incubate at 65°C for 15 minutes.

4. Add 3μl of RNase enzyme to break down the RNA present in the sample. Now place it at 37°C for for 15 minutes. 

5. Now add 200μl of a protein precipitation solution to the sample to precipitate all the protein and other debris present in the sample. Once added, the centrifuge tube is placed in ice for 4-5 minutes and then centrifuged for 4 minutes at maximum speed.

6. All the debris from the sample is precipitated while the DNA remains in the supernatant. 600μl of the supernatant is now removed carefully and transferred into a sterile centrifuge tube.

7. Add 600μl Isopropanol alcohol slowly to supernatant and invert in several time for the DNA to precipitate.

8. The tube is now centrifuged for 1 minute at maximum speed to completely precipitate the DNA.

9. The DNA is precipitated as the pellet and the alcohol remains in the supernatant. 

10. Remove the supernatant and add 600μl of ethanol to clean the DNA. Mix and centrifuge to precipitate DNA. 

11. Remove supernatant and air dry the DNA precipitate for 10-20 minutes.

12. Add 100μl of DNA rehydrating solution to bring the DNA into solution. Mix and place it in a water bath  at 65°C for 45 - 60 minutes.

 The DNA sample thus extracted can be cooled for use in PCR or can be stored at -20°C for future use.

DNA Extraction from Specimens

DNA can be isolated from any living organism. Specimens collected can be processed using the following steps to extract DNA from them. The extracted DNA can be amplified using PCR or further processed to yield the DNA barcode.

 Step 1: Isolation of Sample from the Specimens

 Step 2: Sample Processing for extraction of DNA

 Step 3: Precipitation and removal of proteins and DNA precipitation

 Step 4: Purification of extracted DNA and Elution

DNA PCR Sequencing

In vitro amplification of a DNA sequence can be achieved by Polymerase Chain Reaction or PCR. The requirements for a PCR are, the DNA template strand to be duplicated, specific DNA primers, nucleotide bases, DNA Taq polymerase and a thermocycler in which the reaction will occur. The reaction consists of the following steps: 1. The DNA sequence to be amplified is identified. 2. DNA primers are designed such that they provide 3' ends for replication of the template using DNA polymerase. 3. The DNA to be amplified is heated to cause strand separation. 4. The system is cooled to permit addition and adhesion of the primers to the respective strands. 5. Hybridization of the DNA occurs in the presence of DNA polymerase and the nucleotide bases. At the end of cycle 2 molecules of DNA are obtained from a single DNA molecule. A detailed explanation of the process and an animated version can be seen in the following videos.

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