cDNA and primer Design Help - (Nov/15/2008 )

Hi everyone, I have done my search on this in these forums and it seems that still after 4 hours of reading I'm still a bit confused on this matter.

I did the Blast on the Mus musculus amyloid beta gene and in order for me to design the primer I need to click the CDS button to align the sequence from the staring codon with ATG. So now I need to design the primer! My question is, when designing a primer, is this cDNA sequence going from 5'--->3' and therefore in order to design the primer I have to base pair the primer from the 3' end so that 5'starts with the primer I'm designing???

Also, the primer must have a Tm of at least 68-72 degrees and gc% of 50%.

For example: I need to highlight the primer positions and write the primer sequences and Tms for the primers which I'm going to use for the PCR.

atggcggcca ccgggaccgc ggccgctgcg gccaccggca agcttcttgt cctgctgctg
ctcgggctca cggcgcccgc tgcggctctg gctggctaca tagaggctct tgcagccaat
gctggaacag gcttcgctgt tgctgagcct cagattgcga tgttctgtgg gaagctgaat
atgcatgtga acattcagac tggcaaatgg gagcctgacc caacaggcac caagagctgc
cttggaacaa aggaggaggt tcttcagtac tgccaggaga tatatccaga gctgcagatc
acaaatgtga tggaagcaaa ccagccagtc aatattgata gttggtgccg aagggacaaa
aggcagtgca agagtcacat tgttatacca ttcaagtgtc ttgtgggtga atttgtaagt
gatgtcctgc tagttccaga taactgccag tttttccacc aagagcggat ggaggtgtgt
gagaagcacc agcgctggca cacgttagtc aaggaggcat gtctgactga ggggctgacc
ttatatagct atggcatgct gctgccctgc ggggtagacc agttccatgg caccgagtat
gtgtgctgcc ctcagacaaa gactgttgac tcggactcga ctatgtccaa agaagaggag
ccagaagagc gtcacctgaa caagatgcag aaccatggtt atgaaaaccc aacctacaaa
tacc[b]tggagc agatgcagat ttaa[/b]

Do I need to start with red or the blue?
would the blue be the lower primer and therefore it would be TTAA ATCTGCATCA GCTCCA
Tm= 56 degrees.

I don't know if I'm doing this right.

I'm open for any suggestions

I would appreciate your help..

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If your desire is to PCR out the entire coding sequence, then the red would be your forward primer and the blue would be your reverse primer (written in antiparallel as you have done below the sequence). If you were to use the blue primer, I would probably add two more GG to the end. Whenever you make a primer it is good to have a C or G on the end because it will anneal better to your template (G/C base pairs are stronger than A/T). Adding the two GG's would also help get you closer to a 50% GC content, which unfortunately would be hard to achieve with this sequence.

For future reference, whenever a CDS sequence is given to you it is in 5'-3'order - when you order primers they should be written 5'-3' as well, as you did above. The exception you might find to this would be if you were looking at, say, a sequenceviewer where they show several genes next to each other on a chromosome and some genes are read on the plus strand and some on the minus strand. In that case, they usually have arrows which will show you which strand the gene is located on. If you remember that coding sequences always start with an ATG then you shouldn't get confused.

Hope this was helpful,
smu

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Ok, so then I would need both the forward and the reverse primers for the PCR, right?? In this case however, the red and blue wouldn't work since the Tm is not 68-72 degrees (since thats what i am required to have). So then, I need to go to other sequences in this cDNA and look for a forward and reverse primer sequence that will give me a 68-72 degree Tm and with higher GC codons?

For instances since the Tm for red is 68 degrees this one would work for the forward primer, but the blue sequence (reverse) wouldn't since the Tm is 56 degrees. So then I would need to move along and come up with another sequence in the cDNA or do I just add codons??

i really really appreciate your help.

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It really depends on what you need the cDNA for. If you want the full coding sequence, then you would have no choice but to use the red and blue primer that you highlighted (assuming that the sequence you presented is the full coding sequence). If you just need part of the cDNA then you can look for other primers that fit your primer design requirements. Tm between 68-72 is a probably a guideline for what would be optimal. If you can't follow these restrictions and get what you want, then you need to relax the restrictions. It may or may not be more difficult for PCR purposes. I usually aim for a 50% GC content between 18-26 bases and don't worry too much about Tm, but that's just me. Sometimes I have to fiddle around to get the PCR to work, but it usually isn't too difficult. Adding codons doesn't necessarily help because the purpose of following the Tm guidelines is so that they anneal efficiently to your template. If you're adding arbitrary codons, that sequence isn't there in the cDNA so it doesn't help with the annealing process.

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OK, this is the whole cDNA sequence:

1 atggcggcca ccgggaccgc ggccgctgcg gccaccggca agcttcttgt cctgctgctg
61 ctcgggctca cggcgcccgc tgcggctctg gctggctaca tagaggctct tgcagccaat
121 gctggaacag gcttcgctgt tgctgagcct cagattgcga tgttctgtgg gaagctgaat
181 atgcatgtga acattcagac tggcaaatgg gagcctgacc caacaggcac caagagctgc
241 cttggaacaa aggaggaggt tcttcagtac tgccaggaga tatatccaga gctgcagatc
301 acaaatgtga tggaagcaaa ccagccagtc aatattgata gttggtgccg aagggacaaa
361 aggcagtgca agagtcacat tgttatacca ttcaagtgtc ttgtgggtga atttgtaagt
421 gatgtcctgc tagttccaga taactgccag tttttccacc aagagcggat ggaggtgtgt
481 gagaagcacc agcgctggca cacgttagtc aaggaggcat gtctgactga ggggctgacc
541 ttatatagct atggcatgct gctgccctgc ggggtagacc agttccatgg caccgagtat
601 gtgtgctgcc ctcagacaaa gactgttgac tcggactcga ctatgtccaa agaagaggag
661 gaagaggaag aggatgaaga ggacgaagag gaagactatg atcttgataa aagtgaattt
721 cctactgaag cagatttgga agacttcaca gaagcagcag cagatgagga agaagaggat
781 gaggaggaag gggaggaagt ggtggaagac cgtgactact actatgaccc ctttaaagga
841 gacgattaca atgaggagaa tccaaccgaa cccagcagcg agggcaccat ttcagacaag
901 gagattgttc acgatgttaa agttcccccg actcccctgc caaccaatga tgttgatgtg
961 tattttgaga cctcagcgga tgataatgag cacgcccgct tccagaaggc taaggaacag
1021 ctggaaattc gacatcgaaa ccgaatggac agggtaaaga aggaatggga agaggcagaa
1081 cttcaagcca agaacctccc caagacagaa agacagaccc tcatccagca tttccaagct
1141 atggttaaag ctttagagaa agaagcagcc agtgagaagc agcagcttgt ggaaacccat
1201 ctggcccgag tagaagccat gctgaatgac cgccgacgca tagctctgga aaattacctg
1261 gctgccctgc agtctgaccc acctcggcca catcgcattc ttcaagctct tcgtcgttac
1321 gtccgtgctg agaacaaaga tcgcctgcat accattcgtc attaccaaca cgtgttggct
1381 gttgacccag aaaaggccgc ccagatgaaa tcccaggtga tgacacacct ccatgtgatt
1441 gaagaaagaa ggaaccaaag tctttctctt ctgtacaaag ttccttatgt tgctcaagaa
1501 attcaagagg aaattgatga gctccttcag gaacagcgag cggatatgga ccaatttacc
1561 tcctccatct cagagaaccc tgtggatgtc cgggtgagct ctgaggagag tgaggagatc
1621 ccgccgttcc accctctcca tcccttccca tccttgtctg agaatgaaga cactcagccg
1681 gagttgtacc acccaatgaa aaaaggctct ggaatggcag aacaagacgg gggactgatt
1741 ggtgcagaag aaaaagtgat taacagcaag aataaaatgg atgaaaatat ggtcattgac
1801 gagactctgg atgttaagga aatgattttc aatgctgaga gagttggagg ccttgaggaa
1861 gagccggaat cggtgggacc tttaagggag gatttcagtt tgagcagcaa tgcccttatt
1921 ggcttgctgg ttatcgcagt ggccattgct acggtcatcg ttatcagcct ggtgatgctg
1981 aggaagaggc agtacggcac catcagccac gggattgtgg aggttgaccc aatgctcacc
2041 ccagaagagc gtcacctgaa caagatgcag aaccatggtt atgaaaaccc aacctacaaa
2101 tacctggagc agatgcagat ttaa


I am to highlight the primer positions on the sequence and write down the primer sequences and Tms for the primers which I need to use for the PCR.

Then, I need to clone the gene into expression vector (eukaryotic and/or prokaryotic); and then need to validate cloning and the expression of the protein.

My question is, I would need both the red and blue sequences for the cDNA primers to to be incorporated into the vector by priming the red cDNA onto a start portion of a vecor and the blue on the end of the vector?

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I'm sorry but I don't understand your question. If you want the full length protein, the you take a forward primer starting with the ATG and a reverse primer ending with the stop codon (unless you intend to add a tag to the protein, in which case you should eliminate the stop codon). To clone it into an expression vector, you would probably want to add some restriction sites to the end of the primer, then cut the vector and your PCR with these sites to get the PCR product into your vector.

You should see if there is someone in your lab (or a lab nearby) who can help you with this since you seem to be new to PCR and cloning - getting all of your questions answered by the forum will be less than adequate and not to mention time consuming. Most people take a few hours to days to get back to you and you're wasting time by not seeking out help from someone nearby. Sorry if I sound like I don't want to help you, its just that you'll learn a lot more by seeking out help from someone that you can talk to face to face.

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Trust me I do appreciate all the help I'm getting, and I definitely know what you mean about "seeking help", its just that I've tried that and the lab coordinator isn't the best at speaking English and its very hard to know what she's saying if you know what i mean.

But thanks allot for your help, at least now I know how to start.
thanks again

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