• Mr. Fatih is a cotton breeder in an institute of Ministry of Agriculture, Food and Livestock, Turkey. While evaluating germplasm, he suspects that one cotton variety is BT (Bt Cotton) with no any history (an unknown source). Keeping in view budget constraints, he can detect it only with PCR method. How can he procced ahead to confirm it?
To confirm if the cotton variety is Bt cotton using the PCR method, Mr. Fatih can follow these steps:
1. Get a sample: Mr. Fatih needs to obtain a sample of the suspected Bt cotton variety for analysis. This could be a leaf sample or any other tissue sample from the plant.
2. Extract DNA: The next step is to extract the DNA from the sample. There are various DNA extraction kits available in the market that provide easy and efficient methods for DNA extraction. Mr. Fatih needs to follow the instructions provided with the chosen kit to extract DNA from the sample.
3. Design primers: Primers are short DNA sequences that bind to specific regions of the target DNA sequence. In this case, Mr. Fatih needs to design primers that will specifically bind to the Bt gene in Bt cotton. This can be done using specialized software or by consulting with experts in molecular biology.
4. PCR amplification: Once the primers are designed, Mr. Fatih needs to perform a PCR (Polymerase Chain Reaction) amplification. PCR is a technique that allows the replication of a specific DNA segment. By using the designed primers, he can selectively amplify the Bt gene if it is present in the sample. The PCR reaction requires a DNA polymerase enzyme, nucleotides, and appropriate buffer conditions.
5. Gel electrophoresis: After PCR amplification, Mr. Fatih needs to analyze the PCR products to determine if the suspected Bt cotton variety contains the Bt gene. Gel electrophoresis is a commonly used technique to separate DNA fragments based on their size. He should load the PCR products onto an agarose gel and electrophorese them. If the PCR product matches the expected size of the Bt gene fragment, it confirms the presence of the Bt gene in the sample.
6. Compare with positive control: To validate the results, Mr. Fatih should include a positive control in the PCR analysis. A positive control is a known Bt cotton variety that contains the Bt gene. By comparing the PCR product of the suspected variety with the positive control, he can confirm if it is indeed Bt cotton.
It's important to note that this process requires specialized laboratory equipment and expertise. Therefore, Mr. Fatih may need to collaborate with a molecular biology lab or consult with experts in this field to perform the PCR analysis accurately.