You can do it by two ways

1. 3' prime and 5' race PCR followed by end seuquencing and designing primers for full length PCR amplification. and sequencing

2. Another option is sequencing of PCR product amplified by degenerate primers and the designing primers at 3' and 5' end for end sequnencing and assebling it to form single contig which can used used to predict complete coding sequence by using translate option from swissprot.

good luck

Thank you Sachin for your answer. I am sequencing the PCR product now .

Hi Harshraj,

Just curious,

1. you mentioned "Multiple Sequence Alignment of three genes". Are these 3 genes from 3 different species?

2. If the gene you are working a unique single gene in the genome? 

Three genes which i used for "Multiple sequence alignment" are from rice, barley (Hordeum vulgare) and Phyllostachys edulis. These three plants belongs to poaceae (grass) family. Now after designing the degenerate primer I am trying to isolate the same gene from another plant which belongs to poaceae (grass) family.

I think gene is unique gene (not sure). 

1. 5' RACE and 3' RACE ( Rapid Amplification of cDNA Ends) are useful  tools to find out the ends of partial cDNA as suggested by Sachin. The 3' RACE seems simpler, need an oligo dT primer (see attachment for a summary of these methods).

2. 5'/3' RACE kits are commercial available; such as this one from FishTermo Scientific company:  ( https://www.thermofisher.com/order/catalog/product/18374058 ).

3. Attached is an instruction booklet which details the principle and procedures of 5' RACE on page 3.

4. If you don't want to use a kit. Please see this discussion here on RG for some suggestions:

https://www.researchgate.net/post/Advice_on_5_RACE_without_a_kit

Thank you Yuan for your answer. and in detail explanation.

RACE PCR looks bit tedious process (If I am wrong please correct me). I am thinking to try the second process mentioned by Sachin. What I am going to do is " I have already amplified 8kb region of my gene(size of my gene is 1.2kb). So after sequencing I am thinking to design the primer again using 3' and 5' end sequences and then just assemble the fragments to make the complete gene. do you think this will work better than RACE?

what is your opinion on this?

Waiting for your response.

Thank you !

I know you now have 'partial' cDNA of your target gene, and you are sequencing it. I am not clear about what you said "I have already amplified 8kb region of my gene (size of my gene is 1.2 kb)". You meant that your gene full-length cDNA is 1.2 kb? What is the 8 kb region about? Could you elaborate it for me?

Sorry it  was typing mistake its 0.8Kb

Thanks for the explanation.

So, you have got part of the gene (0.8 kb) from your gene-of-interest (1.2-kb). After sequencing, you will obtain the DNA sequence of this 0.8-kb. Gene-specific primers (GSP) then can be designed from the 0.8-kb DNA. By using one GSP and oligo dT, probably you can easily synthesize the unfished 3'-end and assemble them. 

By using the #2 method mentioned above, I am wondering what is your plan to get the unfinished 5'-end DNA based on 'cDNA' product? How will you design the primer outside the 0.8-kb DNA of the 5'-end? Or, are you planning on using other method based on 'genomic' DNA of this gene? I would like to know your plan to discuss.

Have a good weekend.

Thank you Yuan for detailed explanation and sorry for late response from my side actually I was waiting for sequencing results. In sequencing I got the 867 bp sequence.

My plan to get the unfished region from 3'-end is by using the GSP and Oligo dT and to isolate the 5'-end unfished region I am going to perform the 5' RACE PCR by using the 5'-Full RACE Core Set of TAKARA.

Hi Harshraj,

Good to hear from you. Yes, I think that is the best way to go!!

I just found out that Sigma and Takara/Clontech companies sell these kind of kits for around $600 (US) for 10 reactions. That is expensive!! Sometimes, I think, in some way, science has become rich man's game...

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Just for your future information, you can also use 'genomic DNA' (introns involved) as material to finish the unfinished business. But, still, it is no easy way out. Plenty of work. Below is some of the methods.

[1] Using inverse PCR (IPCR). See the attached picture (attachment 1) for the summarized method for using IPCR. One example using carrots as material as shown (attachment 2). This method involved gDNA isolation, restriction digestion, ligation and PCR. It is a tedious work, not simple.

[2] Using TAIL PCR. The original TAIL-PCR was published in 1995 (see attachment 3). Later, improved version of TAIL-PCR such as hiTAIL-PCR was created (see attachment 4, hiTAIL-PCR).

[3] Using 'Sequecing capture'. A newer method (see attachment 5).

[4] Using FPNI-PCR. See attached paper (attachment 6).

Thank you so much Dr. Yuan for your kind help.

I will read all those things and I will decide what to do. I will update the things here during my experiments.

Good luck to your experiments

Thanks and regards

Harshraj

Hi Sachin,

I'm curious about your suggestion # 2. Is it necessary cloning or ligase treatrament?