Authors: Dr. Monika Singh, Ms. Sushmita (Co-author)
Genetically modified (GM) crops, commonly referred as transgenic or biotech crops are the plants in which the genetic material is altered for desired trait(s) employing tools of recombinant DNA technology or genetic engineering. For studying the expression of transgenes, endogenous reference genes, also known as housekeeping genes, are being commonly employed, as constant reference to normalize the expression of target genes in different tissues and under different conditions.
With the commercial approval and adoption of genetically modified (GM) crops exhibiting agronomically important traits, better nutritional quality and other superior traits, global area under GM crops has been expanded. To check the integrity of a GM crop, detection of specific genetic elements being present in the inserted transgenic construct is required. Prior to testing the GM status of a seed/sample, amplifiability of test samples needs to be confirmed so that chances of false negatives, if any, may be eliminated. Endogenous reference genes are being employed for this purpose.
An endogenous reference gene is a taxon-specific DNA sequence, which may be included as an experimental control in a qualitative polymerase chain reaction (PCR) assay, to check the PCR inhibitors that may hinder successful amplification of target DNA. Major criteria for selection of a taxon-specific gene to be used as an endogenous gene (Ding et al., 2004) include:
- - Species specificity,
- - Single or stable low copy number,
- - Low intra-specific variability of nucleotide sequences.
Besides confirming the amplifiability of test samples and being employed as an internal control, endogenous reference genes also play key role in quantification of genetically modified organisms (GMOs). For quantification, copy numbers of GMO-specific DNA and an endogenous reference gene are measured by real-time PCR and then percent GMO content is calculated as the ratio of these copy numbers. Therefore, selection of an appropriate endogenous reference gene is essential for development of detection methods for GM crops.
Validated real-time PCR protocols detecting specific taxon-specific genes are available at http://gmo-crl.jrc.ec.europa.eu/gmomethods/ .
Methods for detecting endogenous reference genes
- Conventional PCR for detection an endogenous gene to check amplifiability of DNA samples prior to GMO testing;
- Multiplex PCR for simultaneous detection of endogenous gene as an internal control and transgenic elements;
- Real-time PCR for quantification of GMOs or to determine GMO copies.
1) Ding J, Jia J, Yang L, Wen H, Zhang C, Liu W, Zhang D (2004) Validation of a rice specific gene, sucrose phosphate synthase, used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes. Journal of Agricultural and Food Chemistry, 52 (11): 3372-3377
About Author / Additional Info:
We both are currently working as Scientist at ICAR-National Bureau of Plant Genetic Resources, New Delhi.