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👩🔬Let's Design a Primer👨🔬
Primer Design for PCR🧬
Oligonucleotide primers are necessary when running a PCR reaction. One
needs to design primers that are complementary to the template region of
DNA. They are synthesized chemically by joining nucleotides together.
One must selectively block and unblock repeatedly the reactive groups on
a nucleotide when adding a nucleotide one at a time. The main property
of primers is that they must correspond to sequences on the template
molecule (must be complementary to template strand). However, primers do
not need to correspond to the template strand completely; it is
essential, however, that the 3’ end of the primer corresponds completely
to the template DNA strand so elongation can proceed. Usually a guanine
or cytosine is used at the 3’ end, and the 5’ end of the primer has
stretches of several nucleotides. Also, both of the 3’ ends of the
hybridized primers must point toward one another.
The size of the primer is very important as well. Short primers
are mainly used for amplifying a small, simple fragment of DNA. On the
other hand, a long primer is used to amplify a eukaryotic genomic DNA
sample. However, a primer should not be too long (> 30-mer primers) or
too short. Short primers produce inaccurate, nonspecific DNA
amplification product, and long primers result in a slower hybridizing
rate. On average, the DNA fragment that needs to be amplified should be
within 1-10 kB in size.
The structure of the primer should be relatively simple and contain no
internal secondary structure to avoid internal folding. One also needs
to avoid primer-primer annealing which creates primer dimers and
disrupts the amplification process. When designing, if unsure about what
nucleotide to put at a certain position within the primer, one can
include more than one nucleotide at that position termed a mixed site.
One can also use a nucleotide-based molecular insert (inosine) instead
of a regular nucleotide for broader pairing capabilities.
- Length of 18-24 bases
- 40-60% G/C content
- Start and end with 1-2 G/C pairs
- Melting temperature (Tm) of 50-60°C
- Primer pairs should have a Tm within 5°C of each other
- Primer pairs should not have complementary regions
💻Coded by:
👩🏻💻Mahdie Matin
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