PCR is a shorthand, simple but very useful procedure in molecular biology called the polymerase chain reaction. It is a technique used to amplify a segment of DNA of interest or produce lots and lots of copies.
A primer is a short nucleic acid sequence that provides a starting point for DNA synthesis. In living organisms, primers are short strands of RNA. A primer must be synthesized by an enzyme called primase, which is a type of RNA polymerase, before DNA replication can occur.
Primer is designed in 5’ to 3’ direction. Primers are oligonucleotide DNA sequences that are added in the PCR reaction mixture to identify and bind to their specific complementary base sequence in the heterogeneous collection of DNA molecules.
Primers are required for DNA amplification as replication enzymes cannot synthesize DNA de-novo i.e., it requires a 3’ OH to extend which is contributed by the primers.
Two sets of primers called the forward and the reverse primers are used both run from 5’ to 3’ direction but their binding position differ
NOTE: The sequence, received by databases of genes is in 5’ to 3’ direction
We call primer 1 as a forward primer as it is read in the same frame as the original 5’ to 3’ sequence primer 2, however, is a reverse primer complementary of the original 5’ to 3’ sequence.
PROPERTIES OF PRIMER:
- Primer length: optimally 15-23nt, long enough for specificity and short enough to bind easily during annealing
Short primer = ectopic hybridization
Long primer= slow annealing
Note: occurrence of a specific sequence of length ‘n’ is 1/4th i.e. if primer size is reduced frequency of occurring of that sequence to be found increases.
- Primer melting temperature (Tm): the temperature at which half DNA duplex denatures, 52-58°C best result not beyond 65 degree Celsius.
- The annealing temperature of primer (Ta): Temperature at which DNA strand renature (near to Tm), Ta should be optimum 60 degree Celsius to low Ta, Ta = Tm ± 5°C.
- GC content: Should be 40-60% (optimally 55%) G forms triple hydrogen bonds with C, higher GC contents means more hydrogen bonds with means a higher Tm and a higher Ta as well as will make complete denaturation difficult, applying a temperature higher than 95°C might have some effects on the efficiency of Taq polymerase.
- GC clamp: GC at least 5 base at 3’ end promotes specific binding however not more than 3G/C should be added.
- Primer secondary structures: primers can form secondary structures that can lead to poor or no field eg. Hairpin (intramolecular) occurs due to the presence of internal symmetry elements in the primer. Eg: palindromic sequences.
- Repeats: Dinucleotides occurring consecutively, non-specific, and binding occurs should be avoided.
- Run: a long repeat of a single base should be avoided.
- Cross homology: should be avoided by primers
(i). primers should not have binding homology to another gene in the same system.
(ii). Primers should not have binding homology to genes of different individuals, species, similarity to common genes eg. Housekeeping genes should be always avoided.
Designing a primer
- Find the sequence of the template on https://www.ncbi.nlm.nih.gov/
- From available databases.
- From sequencing methods.
- From RNA sequences.
- From protein sequences.
Here I take Homo sapiens p53 genes
2. After finding the sequence from database, search for exons from FASTA where you only get the nucleotide sequence and not the numerical values.
3. Copy and paste the exon region in primer 3.0input and select humans if your sequence is in human. Should select a hybridization probe required for real-time PCR. In
In this you will also get the additional primer sequences
Set the conditions like at general primer picking condition
|Primer GC percentage||40||60|
internal oligo because I am going to design for my probe to non-florescent quencher
|Internal oligo size||18||22|
|Internal oligo Tm||58||62|
|Internal oligo GC percentage||40||60|
4. others condition are preselected in this software than select pick primers.
5. Analyse the primers and pick the best primer suitable for the experiment.
6. You can check whether this primer matches with the human genomic database and click on nucleotide blast on website blast.ncbi you see some sequences are 100% to this primer in the human genome.
The purpose of PCR primers is to provide a “free” 3′-OH group to which the DNA polymerase can add dNTPs. So that we can add new nucleotides and amplify our required gene.
PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides in length. Two primers are used in each PCR reaction, and they are designed so that they flank the target region (a region that should be copied).
Avoid having complementary sequences within the primer or between the forward and reverse primers which usually lead to self-dimers or primer-dimers. As the primer concentration is much larger than template concentration in a reaction, the formation of self-dimers and primer-dimers will seriously compete with template-primer annealing and affects product yield.
To check whether the correct target DNA fragment has been amplified, gel electrophoresis is a quick method to examine the molecular sizes (in bp) of the amplified products. The size(s) of the amplified PCR product is estimated by comparing it to a DNA ladder, a molecular weight marker which contains DNA fragments of known sizes.
As you can see this primer designer technique is so easy to do and less time consuming in software whereas in traditional days primer designing technique used by researchers across the globe in different experiments like cloning, Sanger sequencing, PCR, etc.