Re: [DIYbio] Designing primers for PCR

On Fri, Feb 8, 2013 at 12:47 AM, Mega <masterstorm123@gmail.com> wrote:
> Hi everyone,
>
>
> I'm trying to design primers on my own. but now there are a few questions
> that Google couldn't answer me.
>
>
> a) are primers single stranded DNA or double stranded?
> All results from Google don't say anyhing about that. It's just like,
> everyone who writes thos pages knows, but it is too obvious to be mentioned.

It's explicit when you understand the history (the etymology, where
the name comes from), what comes to my mind is primase and okazaki
fragments...
http://en.wikipedia.org/wiki/Primase
http://en.wikipedia.org/wiki/Okazaki_fragments

" the coding strand is the DNA strand which has the same base sequence
as the RNA transcript produced (although with thymine replaced by
uracil)"

> ATG-AAA-NNNNNNNNNNNNNNNNNN-TTT-TAG

Since mRNA is read from 5' to 3' (5' OH is the terminus  of the
'beginning' of the mRNA where the first codon is), then you know they
coding sequence will have 5' before the ATG and 3' after it

>
> If double stranded Iwould just have to write down the sequence:
>

primers are single stranded

> forward primer:
> NNNrestriction - ATG - AAA
>

this would be 5 'NNNrestriction - ATG - AAA  3' (this will prime the
template strand, and polymerase will add nucleotides to the 3' end)


> reverse primer:
> TTT-TAG-NNNrestriction.site

no, you need to change the bases to their pair

5' etis.noitcirtserNNN-CTA-AAA 3'

or

3' AAA-ATC-NNNrestriction.site 5'

--
-Nathan

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