Primers in molecular biology are used as a start point in DNA synthesis, in vitro as well as in vivo.
The DNA primer is used in PCR amplification while the RNA primer is the main ingredient of replication.
A lot of content is available online on both topics, yet by comparing the DNA primer with the RNA primer, we can understand it very well.
But before that,
Let’s start with the basics,
DNA replication is required for copying the genetic material inherited in daughter cells. The process of replication is an enzyme-dependent catalytic reaction, utilizing many proteins to do so.
On the other side,
PCR is also used for synthesizing DNA but it is a temperature-dependent process. It amplifies millions of copies of DNA for the purpose of research.
The main purpose of both processes is to synthesize new DNA.
Both processes required nucleotides, DNA polymerase and primer as main ingredients for copying DNA.
Read related articles:
However, several other chemicals and ingredients are also required for amplification through PCR.
The DNA polymerase performs one of the important functions in synthesizing DNA, it adds the nucleotides to the growing polynucleotide chain.
Interestingly, the polymerase used in the PCR is not a normal one, it is a special type of enzyme that is temperature stable, called Taq DNA polymerase.
It works even at a higher temperature. Read more on Taq DNA polymerase: Function of Taq DNA polymerase in PCR
But for elongating the polynucleotide chain, every polymerase required a short stretch of a single-stranded nucleic acid which provides a free 3’ OH group.
Functionally, the DNA polymerase is different from the RNA polymerase. Because the DNA polymerase required a starting point, a free 3’OH end from where it starts polymerization by forming a phosphodiester bond.
In the PCR the free 3’ OH group is provided by the DNA primer.
In the present article, we are comparing these two different types of primers used in DNA synthesize.
RNA primer:
First of all, the RNA primer is non-significant for the PCR amplification. However, it is necessary for the replication process.
The RNA primer is a short stretch of nucleic acid made up of the single-stranded RNA molecule.
An RNA polymerase, called DNA primase synthesizes a short stretch of single-stranded RNA molecule for starting replication.
It is very essentially required for a DNA polymerase to start its catalytic activity.
The single-stranded RNA primer provides a free 3’ OH group which is required for DNA polymerase. See the Image,
The RNA primers are of two types used in replication. One, that starts DNA replications and is approximately 10 to 18 nucleotides long, at the leading strand.
While others are used for the synthesis of Okazaki fragments and these are 8 to 10 nucleotides long, at the lagging strand.
Why replication is dependent on RNA primer instead of a DNA primer?
The answer is here,
The only RNA polymerase is available to synthesize the ssRNA thus not DNA but the RNA is used in the replication.
And in the end, due to the exonuclease activity of the DNA polymerase, the RNA primers are removed, simultaneously the gap is filled by the polymerase with the complementary nucleotides and sealed with DNA ligase.
For doing this, the DNA polymerase trackback and finds the RNA primer which is actually not a part of our DNA strand.
(DNA ligase fills the gap between adjacent nucleotides by forming the phosphodiester bond.)
The 3’ to 5’ exonuclease activity of DNA polymerase removes it.
Note: only a single type of RNA primer is used for DNA replication.
Interesting fact:
The DNA polymerase can elongate the polynucleotide strand but can not synthesize it directly (it needs a free 3’ end).
Only RNA polymerase can do so, thus, RNA primer is used in replication.
There is a difference between synthesizing and elongating nucleotide chains, see the image below,
DNA primer:
Like the RNA primer, the DNA primers are also used for the synthesis of DNA.
The artificially synthesized DNA primers are used for DNA amplification during the PCR reaction.
It is a single-stranded molecule of DNA ranging from 12 nucleotides to 25 nucleotides.
Here, the RNA primers can not work efficiently because it is less stable than the DNA primers.
Notably, a pair of DNA primers, one for sense strand DNA called forward primer and one for antisense strand of DNA called reverse primer, is used for amplification of dsDNA.
Both, forward and reverse primer binds in such a way that the Taq DNA polymerase adds nucleotides inward. See the image,
Criteria to select the DNA primer:
We have covered an entire article on how to design DNA primers for PCR. read it here: PCR primer design guidelines.
Broadly, the DNA primer used in the PCR must have the following properties:
- 12 to 20 nucleotides long (for normal PCR)
- Less than or between 45-50% of GC content
- Less hairpin forming capacity
- Annealing temperature between 55 to 65°C
Note:
We are using the DNA primers only for amplification purpose, thus no need for proofreading or exonuclease activity.
The DNA primers are more stable than the RNA primer, even, it can not degrade at a higher temperature.
The polymerase…
The main function of a primer is to provide a junction or substrate to work DNA polymerase and do polymerization.
Although the polymerases used in replication, as well as amplification, are different.
The polymerase used in amplification is stable at a higher temperature and does not have the exonuclease activity.
Whereas the polymerase used in the replication can not work at a higher temperature and have the 3’ to 5’ and 5’ to 3’ exonuclease activity.
The exonuclease activity is essentially needed to proofread the entire sequence after the replication and it removes each and every mismatch nucleotides from the newly synthesized DNA strand.
See the image of proofreading:
A quick summary between RNA primer and DNA primer:
| RNA primers | DNA primers |
| Used in DNA replication (in vivo) | Used in DNA amplification during PCR (in vitro) |
| A polynucleotide chain of 10 to 12 oligos | a polynucleotide chain of 12 to 22 oligos |
| Unstable at a higher temperature | Stable at a higher temperature |
| Removed after completion of replication by exonuclease activity | Remains a part of the newly synthesized DNA strand |
| Synthesized by DNA primase | Synthesized artificially |
| A single molecule of single-stranded RNA is used | Pair of single-stranded DNA, one forward primer and one reverse primer are used for two different single-stranded targets. |
| Adenine, guanine, cytosine and uracil is present in the RNA primer | Adenine, guanine, cytosine and thymine are present in the DNA primer |
Read an interesting article: Introduction To Genetics: Definition, History, Applications And Branches
Conclusion:
Primers are a very crucial ingredient in molecular genetic tools such as PCR or DNA sequencing. Primer designing tools like Primer 3 help researchers to design primer efficiently for performing PCR.
Read our PCR primer designing guideline.
nicely explained, thank you