Thanks but I think you misunderestand!As I mentioned gene that is PEPC of sugarcane and orf starts from the same location.Here I can not use softwar for the Forward primer. I needsomeone to exactly read my questions.

But my question is that is primer can start with ATG or not! and what is problem!

My gene of interest is related to Phosphoenol pyruvate caboxylase of sugarcane , I know the orf and the whole genom of this specific gene!

My second question is how I canfind where the gene of my interest is in sugarcane sequence which is 141,182 bp!!!!If you answer me under my question is better to share idea more with others!

Hi Marjan

Firstly, yes primer can also start with ATG, it doesn’t matter, but the sequences which you selected should be around 20 bp based on gene’s sequences you are looking for and many other factors like G/C ratio,……. You can use Biology Workbench software, the software simply design the primer based on sequences that you copy and paste in software. You can also use ORF fonder in NCBI in below link: http://www.ncbi.nlm.nih.gov/gorf/gorf.html

@Darab, hi my problem is not how design problem!!!!!Anyway thanks.

First of all does the gene that u going to express is a single gene in the genome or putative gene?

@ Hadi Putative!

Hello Marjan,

2. I noticed that the genome sequence you refer to is a Chloroplast genome project not a Whole genome project (http://dnaresearch.oxfordjournals.org/content/11/2/93.abstract). This does not contain the PEPC gene you are interested in.

You will not find any 50 'mRNA' nucleotides before the ATG of the ORF. Remember that 'mRNA' refers to the transcribed gene sequence, When you read 'mRNA' in papers or at NCBI it does not refer to any genomic untranscribed regions (UTR), including 5' and 3' UTRs, or introns. The mRNA is ONLY the exons of a gene. What you are looking for is the 50bp of genomic sequence of the 5' UTR.

To obtain any 5' UTR sequence for your gene I would suggest contacting the authors of this project from Brazil. They may not have published the entire genome yet but they many be of assistance in finding the 5' UTR seq for PEPC (http://www.ncbi.nlm.nih.gov/bioproject/12961).

1. As mentioned from other posts you can design primers with an ATG as the 5' of the Forward primer. I suggest you read this attached paper however and consider adding some plasmid specific sequence to the 5' ends of both the Forward Primer and Reverse Primers to facilitate the cloning process. The most useful plasmid sequences to add are Restriction sites such as EcoRV for example but you can add other sequences as well. The paper discusses designing sequences that also include selectable markers etc and is fungi specific (ie; Site specific transformation in plants is very, very difficult, and requires a protoplast transformation protocol, and you are trying to introduce your gene into other species anyway). I'm guessing you will use an Agrobacteria mediated transformation right and you have some target plasmid for this? In your case if you want to introduce a restiction enzyme cut site such as EcoRV, the Forward primer (5'-3') would look like this GAT^ATCatggcgtccgagcggc. Similarly for the reverse primer (5'-3') GAT^ATCcttcacaacaagagtg. The restiction enzyme cut site you use will depend upon your target plasmid you will clone into. Make sure your PEPC sequence doesn't have the same enzyme cut site that you design into your primers.

1. You can use one pair of primers to amplify the entire gene IF your polymerase enzyme is a "High Fidelity" polymerase enzyme. Meaning it can amplify long sequences (up to 10,000bp in some cases). TAQ polymerase is not a good enzyme for this task because it's efficacy is for relatively short sequences (1000bp), and it usually doesn't have endonuclease "proof reading" capabilities. I suggest a PFU type polymerase, or one specific for longer amplifications. If you are restricted to using TAQ or similar then I suggest increasing the extension time (72 C) to at least 2 minutes and splitting up your PEPC into at least 3 PCR reactions. For this process, make sure to design primers that allow for each of the 3 amplicons to overlap by at least 30 bp. The problem is you will end up with 3 amplicons and cloning these together will be frustrating and time consuming. As mentioned int he paper you can use a single PCR reaction to join these together but again you will need a High Fidelity polymerase. Its worth the $ to purchase a High Fidelity polymerase, it will save you time and energy.

Hope this helps

paul

i agree with what Paul had said.

Why u want to introduce PEPC, there are many publications which show that pepc, when introduced in C3 plants, does not increase co2 fixation in C3 plants. ppl are putting all 5 enzymes of C4 cycle in C3 plants these days anf u r after one gene. please let me know.

Dear Paul many thanks for your answer.

I could not open this link"(http://www.ncbi.nlm.nih.gov/bioproject/12961)"

The Chloroplast sequence accession number is accession: NC_006084 http://www.ncbi.nlm.nih.gov/bioproject?Db=genome&Cmd=ShowDetailView&TermToSearch=17857 and the Saccharum officinarum EST project is accession: PRJNA12951. Hope this helps, the link to the Brazilian University in charge is http://www.unicamp.br/unicamp/.

If you are looking to express this gene into the host plant don't forget you are going to need a plant based promotor system. Merely cloning the gene is worthless if you are looking for gene expression. This isn't as hard as it sounds, there are plasmids out there for Agrobacterium that have multiple cloning sites (MCS) with the tra genes needed for Agrobacterium infection.

In this case you will need to match your restriction sites in your PCR product to the MCS of the plasmid. You will also have to take step to make sure the 5' end of the gene is in step with the 3' end of the promotor for the proper reading frame.