9 replies to this topic

[Mad researcher]

How are primers made?

I design a primer using NCBI primer design and then i send it to a company (invitrogen) but i want to know how do they make the primer with the sequence i have sent them. How do they measure the Tm, the %GC, OD and other factors.

[prabhubct]

you froward primer is your sequence as it is from 5' end to 3' end.
your Reverse primer is Reverse complement of your sequence.

T_m = 4(G + C) + 2(A + T) °C

Ions in solutions affect Tm.
For % GC  you have to calculate number of G and C bases, combine them and divide by total number of bases.
O.D. is how much your absorbance of sample is. More the sample DNA more the O.D.
In other factors consideration is given to self priming, primer dimer sequence complementarity as non-desirable.

[mdfenko]

and this wikipedia page tells how they are made:

oligonucleotide synthesis

[ascacioc]

Tm is a bit more complicated calculated (that is the exam version from prabhubct) I use for Tm's this tool:
http://eu.idtdna.com...er/Default.aspx
what I like about this tool is that it gives you homodimers and heterodimers with your other primer...which is cool that you don't have to think too much

[prabhubct]

ascacioc, on 20 September 2012 - 09:42 AM, said:

Tm is a bit more complicated calculated (that is the exam version from prabhubct) I use for Tm's this tool:
http://eu.idtdna.com...er/Default.aspx
what I like about this tool is that it gives you homodimers and heterodimers with your other primer...which is cool that you don't have to think too much

Yes you are right. I also do not calculate Tm by calculation, I do use one of those available tools. But I suppose question was how it is calculated so I presented science behind it.

[ascacioc]

Yes I know that you presented the science behind it. What I meant it: with this tool, after you submit a primer they give you the result + this info (containing all the advanced knowledge references :
MELTING TEMPERATURE ASSUMPTIONS AND LIMITATIONS

DNA/DNA: +/- 1.4ºC (Allawi '97) LNA/DNA: +/- 2.0ºC (McTigue '04, Owczarzy, 2011) RNA/DNA: +/- 2.7ºC (Sugimoto '95) DNA/RNA: +/- 2.7ºC (Sugimoto '95) RNA/RNA: +/- 1.3ºC (Xia '98) Divalent cation correction +/- 0.5ºC (Owczarzy '08) Triphosphate correction +/- 0.0ºC (Owczarzy '08) Monovalent cation correction: +/- 2.0ºC (Owczarzy '04)

• Predictions are accurate for oligos from 8 to 60 bases in length, in neutral buffered solutions (pH 7 - 8) with monovalent cation (Na⁺) concentrations from 1.2 M down to 1.5mM, divalent cation (Mg⁺⁺) concentrations from 600 mM down to 0.01 mM, and triphosphates (dNTPs) concentrations up to 120% of the divalent cation concentration.
  
• Oligo concentration is assumed to be significantly larger (at least 6x) than concentration of the complementary target, which is true in majority of molecular biology experiments. If this is not a case, concentration of the target cannot be ignored and you should enter in the box, Oligo Conc = [strand1] – [strand2]/2 when [strand1] ≥ [strand2] Oligo Conc = ([strand1] + [strand2])/4 when [strand1] = [strand2]
  
• Melting temperature accuracy and models: (Oligo/Template)
  
• Consecutive LNA bases hybridized to a DNA template use a model from Owczarzy '11. In the absence of empirical data, LNA bases on an RNA template assume RNA values, and predictions are therefore less accurate.
  
• Non-consecutive LNA bases hybridized to a DNA template use a model from McTigue '04. Consecutive LNA bases on a DNA template and any LNA bases on an RNA template assume RNA energetic parameters and predictions are therefore less accurate.
  
• Effects of chemical modifications are neglected except when the modification contains a base, e.g., 5-Methyl dC, Internal Fluorescein dT. Energetic effects of these modifications are only approximated.

[prabhubct]

ascacioc, on 20 September 2012 - 09:54 PM, said:

  

Oligo Conc = [strand1] – [strand2]/2 when [strand1] ≥ [strand2]      
Oligo Conc = ([strand1] + [strand2])/4 when [strand1] = [strand2]

Is strand means Strand 1 = primer sequence concentration.
strand 2 = Template concentration ?

or it's two strands formed after denaturation.

Thanks.

[ascacioc]

I think it is about the two strands after denaturation; but this is smth you need very rarely and only if you do weird stuff aka non-PCR

[prabhubct]

No not abt non-PCR. I was just wondering in that formula [strand2]  is alwas less than or equal to [strand1]. [strand2] Never more. just curiosity!

Edited by prabhubct, 21 September 2012 - 11:33 AM.

[ascacioc]

if strand 2 would be more than strand 1, and since strand 2 competes with your shorter primer, your primer would never bind to your strand 1. This is why that case is not taken into account because you don't do it.