Designing oligonucleotides and making sure that you have the right parameters for your oligo is an important step in securing results, especially in PCR Primer Design. In order to achieve successful DNA amplification, it’s important to start off with the right primer.
What makes a good primer?
Here are some guidelines for designing your PCR primers:
- Aim for the GC content to be between 40 and 60% with the 3’ of a primer ending in G or C to promote binding. This is known as a GC Clamp. The G and C bases have stronger hydrogen bonding and help with the stability of the primer. Be mindful not to have too many repeating G or C bases, as this can cause primer-dimer formation.
- A good length for PCR primers is generally around 18-30 bases. Specificity usually is dependent on length and annealing temperature. The shorter the primers are, the more efficiently they will bind or anneal to the target.
- Try to make the melting temperature (Tm) of the primers between 65°C and 75°C, and within 5°C of each other. Because the Tm is dependent on the length, it’s important to keep primers on the shorter end. The bases also impact the Tm, G and C result in higher melting temperatures than A and T. If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.
- Typically, 3 to 4 nucleotides are added 5 ’of the restriction enzyme site in the primer to allow for efficient cutting.
- Try to avoid regions of secondary structure and have a balanced distribution of GC-rich and AT-rich domains.
- Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT).
- Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.
- If you are using the primers for cloning, we recommend cartridge purification as a minimum level of purification.
- If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer.
- If you are using the primers for a PCR reaction to be used in Invitrogen TOPO cloning, the primers should not have a phosphate modification.
For Research Use Only. Not for Use in Diagnostic Procedures.
5. Try toavoidregionsofsecondarystructureandhaveabalanceddistributionofGC-rich and AT- rich domains.
What is the 5th point???
Please edit properly before you upload something.
Try to avoid regions of secondary structure and have a balanced distribution of GC-rich and AT- rich domains.
5. Try toavoidregionsofsecondarystructureandhaveabalanceddistributionofGC-rich and AT-rich domains.
What is the 5th point???
Please edit properly before you upload something.
4. Typically, 3 to 4 nucleotides are added 5 ’of the restriction enzyme site in the primer to allow for efficient cutting.
What does this means please. Can I have an explanation !?
In case of designing cloning primers, 3 or 4 nucleotides are added at 5′ end before the restriction site to facilitate efficient cutting.
Anyone please send the flow chart of primer design for Bacterial DNA diagnosis and Viral RNA diagnosis. Flow Chart means starting to end (Taking a sequence from NCBI to final primer design)
In case of designing cloning primers, 3 or 4 nucleotides are added at 5′ end before the restriction site to facilitate efficient cutting.
Nice work, but please, can you explain point 6 “Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT)”??
Oligos containing these types of sequences are typically more difficult to synthesize and may have non-Watson/Crick secondary structures that could interfere with their performance.
Oligos containing these types of sequences are typically more difficult to synthesize and may demonstrate non-Watson/Crick secondary structures, changing their behavior.
Oligos containing these types of sequences are typically more difficult to synthesize and may have non-Watson/Crick secondary structures that could interfere with their performance.
could any one help me with determining what set of primers go with these PCR reactions:
Set 1: 5’TACACTTATACTTTC3’ and 3’GTAAACCGCGCATTAG5’
Set 2: 5’CTCGAGGTGAATAT3’ and 3’CCGCGCATTAGCTAT5’
Set 3: 5’GAGTTACACTTATAC3’ and 3’TGGCGAGTAATCGATA5’
those already look like primers to me, unless you’re doing 6 PCR reactions
Great info! But I was wondering how primers can be designed in such a way that one of them is synthesised first?
I have a confusion. Can designing a pcr reaction and designing of PCR primers are equal?
PCR primers are just one of the “ingredients” for PCR. Besides primers you will need a polymerase, nucleotides, minerals, ex. and those details, along with the thermocycling temperatures and number of cycles, are what constitute the design of the PCR reaction. There are plenty of protocols you can find online of how to design a PCR reaction based on what you are hoping to accomplish.
which ends of primer should have more CG cnontent? and why?
why do we consider start, stop, melting temperature, gc content, primer length, template strand: plus minus. in designing a pcr primer
If i want to design primers at the program..
this is the DNA sequence that i want to PCR..
5’GAAGAAAGAA CACAAAAAGC AGGAAGGGAA GGGGACACAG
CCAGGTTTGA TGAGCCCAGA CTCTCCATTA AACGCTGCGC
TCAGGGGCCC CAGCTGGGGT CATTGACCCC TGAGCAACAC
TCTCTTAAGC TGGAGGATCT CGCCTTGCAG GTTGAAGAGG
TTACAACACA GAGCAGGGGC TGGGGCCTGG CATGAGGCAC
3’TGATCAATCA GTGCCACAGA TACTGCTGTT CTTAGGATGA
GAGCAGAAGC TGGGGTGTCC CACCACAAGC AGGACAGAGA
GGACACCTCT TTGTCATCAG AGATTAATGG GACCCTTGTC
AGGCCCAGGG TGCAGTGGGG GAAGGGCAGA AGGGACCCTG
CCCTCTCAAA GGCACAGTGA AGCTCTGAAC TGAAGGAGCT
Should I used sequence from 5′ end?
and can i check if the primers resulting the correct sequence?
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Hi,
My Primer length is 42 nucleotides, (main seq. is 30nts, rest is adapters and Recognition seq.)
GC content is~33, Tm= 64, HPLC purified.
Primer seq.- GCGG TTAATTAA ATG GCA AAT TAT AAT TTC AAG TAC ATC GCC
I am not getting any amplification product.
Suggestions please!!
Hi,
My primer length is 42 nts. (main seq.= 30 nts, rest is adapters and Recognition seq.)
GC content= 33, Tm= 64, HPLC purified
Primer seq.- GCGG TTAATTAA ATG GCA AAT TAT AAT TTC AAG TAC ATC GCC
I am not getting gene specific amplification product (probably, primer dimer only)
Suggestions please!!
Hi,
My primer length is 42 nts. (Main seq.30 nts, Rest is adapters and recognition seq.)
GC= 33, Tm=64°C, HPLC purified.
I am not getting my gene specific product (probably, primer dimer only)
Primer seq.= GCGG TTAATTAA ATG GCA AAT TAT AAT TTC AAG TAC ATC GCC
Suggestions please !!
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Really nice content about pcr primer designer tips.Thank You for sharing such a nice post
,thanks for sharing. Good stuff!
Nice job