I am working on the gene which has DNA sequence. However, database do not support full length cDNA (just a part of it can be identified and database announce that ATG can not be detected). How can I determine the transcription start site? Anyone suggest simple way to solve this matter? Thank you in advance.
The only way to determine the 5' TSS with complete confidence is to purify capped mRNA, convert to cDNA, and sequence. The CAGE technology (cap analysis of gene expression) from RIKEN is described here: http://fantom.gsc.riken.jp/protocols/basic.html
To some extent, this technology has been eclipsed by GRO-cap sequencing, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3687364/ but has yet to be applied to rice, to the best of my knowledge.
Annotated TSSes for the rice genome are available at the PlantProm DB, http://www.softberry.com/berry.phtml?topic=plantprom&group=data&subgroup=plantprom
I'd suggest you check for the promoter annotation for your GOI within the PlantProm DB, to see if a TSS has been mapped already.
1. Try blast your partial DNA sequence in NCBI or any database.
2. If there is similarity with other nucleotides of DNA sequences, you will get rough idea where the transcription start site is located.
1. Best choice is to do primer-extension experiment, it is relatively easy but usually you need to use radioactivity. Helps also to find alternative transcription start sites.
2. Do some kind of 5’RACE experiment (based on RT-PCR), at least you will be able to find maybe not exact transcription start site, but approximate 5’ end of the RNA.
3. Do cRT-PCR: circularization of RNA by ligation (“head-to-tail”), then RT-PCR and optional cloning. Sequencing of the clones may help you do find exact 5’ends but also 3’ ends and polyA tail lengths.
4. If you do not want to do wet work… depending of the organism that you work with and available data databases: try to find RNAseq data aligned with loci sequence. You have to remember that some sequences are preferably amplified during library prep and also your loci may be affected of up-stream transcription of non-coding RNAs.
2 and 3, depending of the organism and nature of RNA, may need removal of cap structure from 5’end of eukaryotic mRNA using horribly expensive TAP enzyme.
@ Azzreena: I did check on NCBI database but it only obtain partial cDNA sequence. Thank you so much for your advice.
@Michal Koper: I am very appreciated your suggestions, they may help me a lot in this case.
@Daniel: I am working on rice.
I did clone and sequencing my above desire cDNA. My sequencing sequence is 1740 bp, database sequence (same gene) is 1089. When I took blast my sequencing sequence and database sequence. It give 100% match with match region from 1-1089 of RAB-DB sequence and 652-1740 of sequencing sequence. Do you have any idea about what should I do next?
Thank you so much for your help.
The only way to determine the 5' TSS with complete confidence is to purify capped mRNA, convert to cDNA, and sequence. The CAGE technology (cap analysis of gene expression) from RIKEN is described here: http://fantom.gsc.riken.jp/protocols/basic.html
To some extent, this technology has been eclipsed by GRO-cap sequencing, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3687364/ but has yet to be applied to rice, to the best of my knowledge.
Annotated TSSes for the rice genome are available at the PlantProm DB, http://www.softberry.com/berry.phtml?topic=plantprom&group=data&subgroup=plantprom
I'd suggest you check for the promoter annotation for your GOI within the PlantProm DB, to see if a TSS has been mapped already.
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