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Primer Design GCG Sequence

Primer Design GCG Sequence - PCR - Polymerase Chain Reaction Forum

Primer Design GCG Sequence - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 07-29-2008, 06:55 AM
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Question Primer Design GCG Sequence



Hi guys I got this from Amanda in an email. Could someone please help?
thanks:

I'm Amanda, student of biology and tranning work at plant biotechnology.
Searching in the internet, I discovered this site. tham I would like to do a
question.
In the primer designing, what the importance of GCG sequence In the end the
sequence?
early I thank you.
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Old 07-29-2008, 04:13 PM
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Default Re: Primer Design GCG Sequence

Having a "GC clamp" on the 3' end of the primer helps keep the 3' annealed to the templates for proper elongation by the polymerase. Generally you want to try to have 1 or two G/C's. If you have a run of A's T's at the 3' end, the primer can "flop around" on the end making the reaction less efficient.

More GC's allow you to raise annealing temperatures and thus hopefully get more more specific annealing to the template and not some other similar sequences. Of course you need to balance out the sequence to prevent nonspecific annealing. For example, a run of 5 G's on the 3' end will be a great clamp, but it will result in a lot lot of nonspecific amplification products.

5'-nnnnnnnnnGGCTGGGC-3' would have a high melting temp, but bind any SP1 consensus sequences.

5'-nnnnnnnnnGACTTGGC-3'
5'-nnnnnnnnnGACTTAGC-3'
5'-nnnnnnnnnGACTTGTC-3'
5'-nnnnnnnnnGACTGTAC-3' These 4 examples could be reasonable primer ends depending on the sequence and Tm of the pairs (assuming that sequence is not common in the DNA being amplified)

5'-nnnnnnnnnGGCTGTTAT-3' would probably not be very good if the annealing temp of the primer was high.

Here are some other parameters to look at.
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Old 07-30-2008, 10:48 PM
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Default Re: Primer Design GCG Sequence

السلام عليكم ورحمه الله وبركاته
الرجاء لاهل الخبرة في مجال ال
pcr
وكذلك كيفية استخدام هذة التقنية في التعريف الكائنات البكترية وكذلك عمل لها
sequence
فانا مبتدء في هذا المجال ولا املك غير مساعدتكم في تعلم هذا العلم وكذلك اريد معرفة كيفية عمل تصميم لي primer
وعمل bioinformatics
يا ريت من لدية الخيرة في هذا المجال لا يبخل علين بالرد
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