A good method to identify a gene in any living organism is by the other living organism which is close to the first in taxonomy and then make all the molecular work to find out if that gene does exist in the studied organism.
If sequence information and other literature details are not supporting - go for a random method like RAPD and AFLP first.
Suppose that we are interested in some specific trait of plant (eg. fragrance / drought tolerance), if we don't have good evidence of previous works from which step to which step that we can go, to detect the specific gene for that trait?
What are the available methods to find a gene of interest and it's expression levels?
If you can please recommend reading material to get information on identification of genes / QTL and gene expression with basics?
Thank you
As far as I know you can use 2 different approaches depending on your budget. The more traditional method is called Differential Display (DDRT-PCR or DD-PCR) and you can find several papers in the literature. The most modern approach is to use RNA-Seq or Whole Transcriptome Shotgun Sequencing, which is a technology that uses the capabilities of next-generation sequencing to get a snapshot of RNA presence and quantity from a genome at a given moment in time. You can use 2 individuals or groups from both ends of the distribution in the trait of interest. and once the data are obtained apply bioinformatics tools to compare the expression levels of thousands of genes, many of them non characterized yet. From there you can start playing with possible candidates, characterize them and seek for their function using modern plant transformation technologies.
@ dear Prof. Gonzalo Martínez-Rodríguez, Sir thank you very much for making me aware of new technology, I'll try yo read about this DDRT-PCR.
@ dear Dr.Vijaya Chitnis, sir I just wanna know how to apply molecular biology tools to detect gene of interest if prior information is unavailable. specially in Cereals.
What plant species are you refereing to? I see that Vijaya has also asked the same question. If you don't have a reference genome, the most reliable approach is to do RNA sequencing or genome sequencing using next generation sequencing or both to assemble transcriptome and/or genome. It is better if you could use paired-end (this means that each sequencing fragment is sequences from the both ends i.e. from 5' and 3' ends) approach for sequencing.
RNA sequencing approach:
1. Use velevet, trinity or similar tools for de novo transcriptome assembly.
2. Use tools like blast or blat with plant protein databases as reference databases and your contigs as querry sequences to find homologs.
3. You can also do six frame translation of your contigs (if the sequencing is strand specific you only need to do three frame translation) and use tools like inparanoid to find homologs.
4. Then you can design few primers using your contigs to validate your sequences by Sanger sequencing.
Genome sequencing approach
1. Use assembly tool for de novo genome assembly. These tools can assemble reads into contigs and then scaffolding tools (some assmbly tools have their own scaffolding tools) can make scaffolds using contigs.
2. If you have both RNA sequencing libraries and genome sequencing libraries you can use these libraries to strengthen both transcriptome assembly and genome assembly.
3. You can also map your genome contigs to reference genomes of closely related plant species to structure your genome, but the major issue in this approach is loosing information on chromosomal rearrangements.
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Gene Expression