Question

1. Describe at least one procedure step that is important for reducing the chance of contamination of the PCR reaction and yielding false results.
2. Why is it important to weight the samples before adding them to the mortar?
3. If the plant primers do not generate a PCR product when amplifying the test food, can you trust the data obtained using the GMO primers to amplify the test food?
4. If the GMO primers generate a PCR product with the non-GM control food, can you trust the data obtained using the GMO primers to amplify the test food?

Answer 1

1. One of the major reasons for contamination of PCR sample and obtaining false positive results is carryover of previously amplified DNA or mixing of reagents used   or from culture themselves. Pipetting is the major reason for contamination. It is possible to reduce this type of contamination by using different areas for performing the procedures for preparation of the PCR reaction and also the post-amplification steps. Reuse of pipette tips should be avoided. Aerosol resistant tips and positive displacement pipettes can be used to minimize the contamination. All reagents used should be prepared in sterile water. Pipette tips that dispense the PCR master mix should not be touched to reaction mixture. This is a major reason for contamination and false positive results. Pipette tips should be sterilized prior to use and gloves should be used during pipetting.

2. The chemicals used for extraction of DNA/RNA from the sample have to be added in a specific proportion as compared to the sample amount. Too much of sample will cause decreased extraction while too less sample may harm the sample. Weight the sample will give an indication as to how much is required for the experiment. If there is too much sample, it is possible that the sample will absorb all the liquid added to the mortar and pestle. This will not allow the grinding process to happen properly. Similarly, if the sample amount is smaller, there may be spillage of liquid during extraction, leading to loss of extracted DNA/RNA.

3. The plant primers are used as a control to ensure that the PCR amplification occurs from the plant material and not from any contamination. If the plant primers are not amplifying the product from the test sample, then the DNA extraction from plant material failed. These primers will amplify the gene that is obtained from plant DNA and will be seen as a band after electrophoresis. Amplification from GMO primers and not from plant primers in test food indicates that there is contamination and amplification from non-plant source. Hence, these results cannot be relied upon.

4. The GMO primers will amplify the region that is a part of the construct integrated in the GMO food. The construct is responsible for the attainment of new phenotype by the food as it expressed the gene responsible for genetic modification. For example, the GMO primers generally amplify the 35S promoter region of California mosaic virus that is used to express the foreign gene. Thus, this region should not be present in non-GM food as it is incorporated only in GMO food DNA. Amplification of this region annealed by GMO primers in non-GM food is a false positive reaction. Hence, this data is not reliable as it signifies contamination of non-GM DNA with the GMO DNA.