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Forward and reverse primers are the two main type of primers that are necessary components of polymerase chain reaction, commonly called PCR. The main difference between forward and reverse primers depends on the strand that the primer will bind. Primers are short strands of nucleic acids (DNA or RNA) responsible for DNA synthesis.
- Amplification of the antisense strand is done with the use of forward primer.
- The template strand of DNA plays an essential role in the synthesis of mRNA.
- The strand which is complementary to the sense strand is called an antisense strand.
Forward and Reverse Primer
The clear understanding of comparison between forward and reverse primers is necessary to carry out Polymerase Chain Reaction. In this article we have covered the details of forward and reverse primers with examples.
| Table of Content |
Forward Primers
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The attachment of forward primer takes place at start codon of the template DNA. These primers are responsible for synthesizing upper strand of DNA by considering the bottom strand as a template.
- These primers bind to the antisense or (-) strand of double-stranded DNA.
- The antisense strand runs from a 3’ to 5’ direction.
- The example of forward primer is written as: 3I-ATGGCATCCACACACCAATC-5I.
Reverse Primers
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Reverse primer anneal to the sense or (+) strand of DNA (double-stranded).These primers are responsible for synthesizing lower strand by considering upper strand as a template.
- The sense strand runs from a 5’ to 3’ direction.
- The sense strand is called the coding strand as it is complementary to the template strand.
- The example of reverse primer is written as: 5I-CTAACCACACACCTACGGTA -3I
Differences between Forward and Reverse Primer
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Forward and Reverse primers differ from each other in occurrence, annealing of strands, coding, etc. The melting temperature(Tm) of both primers are decided during the time of primer designing. Usually, the range varies between ±5oC that means if the melting point of forward primer is 55oC, then reverse primer should have Tm of 60oC or 50 oC. The table below highlights the major differences between the forward and reverse primers.
| Forward Primer | Reverse Primer |
|---|---|
| Forward primers are the DNA stretches that are complementary to the antisense strands. | Reverse primers are the DNA stretches that are complementary to the sense strands. |
| Since they occur in the 5’ end of the PCR product they are called 5’ primers. | Since they occur in the 3’ end of the PCR product they are called 3’ primers. |
| They bind to the antisense strand (3’-5’). | They bind to the sense strand (5’-3’). |
| Responsible for amplification of antisense strand. | Responsible for amplification of the sense strand. |
| They anneal with the template strand to initiate the synthesis of the coding sequence. | They anneal with the non-template strand to initiate the synthesis of the non-coding sequence. |
In conclusion, the orientation of DNA strand determines the main difference between forward and reverse primers. In PCR, both types of primers are used to amplify or increase the content of target DNA.
Things to Remember
- Forward primers and Reverse primers are the two types of primers used in biology.
- A primer used in the initiation of DNA replication is called an RNA primer.
- The type of primer used in PCR is called a DNA primer.
- The binding of forward primer takes place at template DNA whereas reverse primer binds to its complementary strand.
- The difference between forward and reverse primer is identified by the orientation of DNA strand.
Sample Questions
Ques. What is a non-coding and a coding strand? (2 marks)
Ans. The DNA strand that is used in the process of synthesis of RNA from DNA is called non-coding or antisense strand. The non-template strand with the same sequence as mRNA strand is called sense or coding strand.
Ques. Is primer annealing the part of PCR? (2 marks)
Ans. Yes, annealing of the primer is one of the important steps in PCR.
- In annealing the primers attach to the DNA sequence which is followed by the amplification process.
- The required temperature for the annealing process is 52-58℃.
Ques. Explain briefly about the steps of PCR? (3 marks)
Ans. The important steps of PCR are:
- Denaturation of DNA- This process involves the breaking of double-stranded DNA at a very high temperature.
- Annealing- After denaturation of DNA, the primers are attached to the DNA template at a temperature of 55℃.
- Extension of Primer- In this process, DNA polymerase called ‘taq polymerase’ is attached to the DNA template to add nucleotides for the formation of a new double-stranded DNA
Ques. What is the length of DNA and RNA primers? (1 mark)
Ans. Number of basepairs in DNA and RNA primers are as follows:
- DNA primer- 18 to 24 base pairs
- RNA primer- 10 to 20 base pairs
Ques. Why are DNA primers used in PCR? Give three reasons. (3 marks)
Ans. DNA Primers are most commonly used in PCR due to the following reasons:
- DNA primers are more stable than RNA primers.
- Can be synthesized easily when compared with RNA primers.
- DNA primers unlike RNA primers can be removed after the completion of reaction.
Ques. What are the types of primer dimers? (2 marks)
Ans. Homodimer and Heterodimer are the two types of primer dimers. When two identical primers bind together, homodimers are formed. Heterodimers are formed when two different primers bind together.
Ques. Name the five components of PCR? (1 mark)
Ans. The five components used in polymerase chain reaction or PCR are a DNA template, a DNA polymerase, primer, nucleotides, and buffer.
Ques. Which enzyme is used in amplification of DNA strands? (2 marks)
Ans. Amplification is a process in which two or more copies of DNA are synthesized.
- This is a two step process in which the first step involves the separation of DNA strands.
- This step is followed by the synthesis of two fresh copies of DNA by the action of Taq. polymerase enzyme.
Ques. What do you mean by a primer dimer? (2 marks)
Ans. The primer dimer is a common problem in PCR which affects the results.
These are unwanted strands of PCR products that are caused by the amplification of primers.
Ques. Is PCR and RT-PCR same or do they differ? (3 marks)
Ans. No, both are different and table below highlights the key differences between the two:
| Polymerase Chain Reaction | Reverse Transcriptase PCR |
|---|---|
| In this case, double-stranded DNA acts as a template. | RNA strand serves as a template for reverse transcription and single-stranded DNA is the template for PCR |
| The enzyme used is DNA polymerase | The enzyme used is RNA polymerase |
| Primers used: forward and reverse | Primers used: reverse |
Ques. What is a reverse transcription? (2 marks)
Ans. Reverse transcription is a process of synthesis of DNA from RNA with the help of an enzyme called reverse transcriptase. RT-PCR or reverse transcription PCR is used in gene expression and diagnosis of infections.
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