3 replies to this topic

[tank1917]

Hello everyone:
I was really confused about my new project. I was concentrate on dairy cow nutrition so I was really new in molecular biology. My professor required me to obtain the cow's full length cDNA of CaBP-D9K, which is a part of his research contents of NSFC (China's NIH). I search this gene on NCBI and find the mRNA complete CDS (Access No: M18344). But I don't know the follow step. Some people told me I just isolate the RNA from cow blood, synthesis the 1st stand of cDNA, then amplify the target sequence, and subsequently connect the vector plasmid. Other people told me use 5’ and 3' race or chromosome walking. I really don't know how to solution this problem, would anyone help me? Thanks a lot!

[bob1]

A full length mRNA molecule is usually composed of 4 major parts, first off there is what is known as a 5' UnTranslated Region (5'UTR), which is anything 5' of the start codon, then there is the coding sequence starting with AUG and ending in one of the stop codons, and then there is the 3' UTR and finally a polyA tail.  Not all of these things will always be present, but they often are.

In your accession you have all 4, as you can see in the information pasted from the accession you listed above:

Quote

FEATURES             Location/Qualifiers
     source          1..411
                     /organism="Bos taurus"
                     /mol_type="mRNA"
                     /db_xref="taxon:9913"
                     /dev_stage="3-week-old calf"
     mRNA            <1..411
     CDS             16..255
                     /codon_start=1
                     /product="calcium-binding protein"
                     /protein_id="AAA30420.1"
                     /db_xref="GI:162775"
                     /translation="MSAKKSPEELKGIFEKYAAKEGDPNQLSKEELKLLLQTEFPSLL
                     KGPSTLDELFEELDKNGDGEVSFEEFQVLVKKISQ"
     polyA_signal    390..395
ORIGIN      
        1 ttccaagaac caaaaatgag tgccaaaaag tctccagaag aactgaaggg cattttcgaa       61 aaatatgcag ccaaagaagg tgatccaaac caactgtcca aggaggagct gaagctactg      121 cttcagacgg aattccccag tttgctgaag ggtccaagca ccctcgatga gctttttgaa      181 gaactagaca agaatggaga tggagaagtt agtttcgaag aattccaggt gttggtgaaa      241 aagatatccc agtgaaggaa agaaaaaaat atcattctca gtactggaag aagagtgcct      301 gggatgtggt cctactctgt acaaaaccac caatgtcacc acttaattct tgacaattgt      361 aatgatccaa cactgaggtc taattactga ataaagaaat cctcaaacat g

The bit where it says mRNA, indicates that this is the coding sequence, and that it is based on the cDNA (note the U's turned to T's).  The actual coding region is indicated by the CDS part (16-255). Anything 5' or 3' of the CDS is UTR.  The poly A tail is indicated in the last line before the sequence.

Edited by bob1, 17 November 2011 - 04:41 PM.

[tank1917]

bob1, on 17 November 2011 - 04:37 PM, said:

A full length mRNA molecule is usually composed of 4 major parts, first off there is what is known as a 5' UnTranslated Region (5'UTR), which is anything 5' of the start codon, then there is the coding sequence starting with AUG and ending in one of the stop codons, and then there is the 3' UTR and finally a polyA tail.  Not all of these things will always be present, but they often are.

In your accession you have all 4, as you can see in the information pasted from the accession you listed above:

Quote

FEATURES Location/Qualifiers
source   1..411
/organism="Bos taurus"
/mol_type="mRNA"
/db_xref="taxon:9913"
/dev_stage="3-week-old calf"
mRNA <1..411
CDS 16..255
/codon_start=1
/product="calcium-binding protein"
/protein_id="AAA30420.1"
/db_xref="GI:162775"
/translation="MSAKKSPEELKGIFEKYAAKEGDPNQLSKEELKLLLQTEFPSLL
KGPSTLDELFEELDKNGDGEVSFEEFQVLVKKISQ"
polyA_signal 390..395
ORIGIN   
1 ttccaagaac caaaaatgag tgccaaaaag tctccagaag aactgaaggg cattttcgaa    61 aaatatgcag ccaaagaagg tgatccaaac caactgtcca aggaggagct gaagctactg   121 cttcagacgg aattccccag tttgctgaag ggtccaagca ccctcgatga gctttttgaa   181 gaactagaca agaatggaga tggagaagtt agtttcgaag aattccaggt gttggtgaaa   241 aagatatccc agtgaaggaa agaaaaaaat atcattctca gtactggaag aagagtgcct   301 gggatgtggt cctactctgt acaaaaccac caatgtcacc acttaattct tgacaattgt   361 aatgatccaa cactgaggtc taattactga ataaagaaat cctcaaacat g

The bit where it says mRNA, indicates that this is the coding sequence, and that it is based on the cDNA (note the U's turned to T's).  The actual coding region is indicated by the CDS part (16-255). Anything 5' or 3' of the CDS is UTR.  The poly A tail is indicated in the last line before the sequence.

Thanks bob1 for u kindly reply.

A friend of mine, focused on animal genetic breeding, suggested me to design 2 pairs of primer, which was used to amplify the up and downstream CDS part,respectively. Then assemble the 2 parts to obtain the cow's CaBP sequence.

In fact, I was still a little confused but i'll try

Would u please recommended me some websites and hand-by-hand tutorials on use NCBI to solve my question?

Thanks very much

[bob1]

You should ask your supervisor if you want the UTRs as well as the actual coding sequence, if you are doing expression experiments where you are cloning the gene into a plasmid, you will probably only want the coding sequence.  Making primers for the coding sequence only is easy - for the forward you start at the start codon and go out 20ish bp and  for the reverse start at the stop codon and come in 20ish bp, remembering to reverse complement the sequence.

PCRing your cDNA should be really easy,  as it is so short.