PCR followed by agarose gel electrophoresis is one of the most common workflows in molecular biology. After amplifying a target DNA region, researchers often run the PCR product on an agarose gel to check whether a fragment of the expected size has been obtained.
However, PCR is not simply a method for making more DNA. By selectively amplifying a defined DNA region, PCR can be used for mutant screening, genotyping, SNP detection, DNA sequencing, cloning, and many other applications.
In this article, we follow the workflow from PCR primer design to agarose gel electrophoresis and explain how DNA fragment size can be estimated from a gel image using a DNA ladder and semi-log calibration.
What does PCR actually do?
PCR, or polymerase chain reaction, selectively amplifies a specific region of DNA. A forward primer and a reverse primer are designed to bind on opposite sides of the target sequence, and the DNA between them is repeatedly copied.
This means that primer design determines which region of the genome or DNA molecule will be analyzed. If the distance between the forward and reverse primers is approximately 700 bp, a PCR product of about 700 bp is expected when amplification occurs correctly.
This predicted size is often referred to as the expected amplicon size.
BioChemCalc provides a PCR Primer Design Tool that can be used to select primer candidates from a DNA sequence and evaluate parameters such as melting temperature, GC content, hairpin formation, and primer dimerization.
What can amplified DNA be used for?
The purpose of PCR is not simply to increase the amount of DNA. Selective amplification makes a specific DNA region easier to detect, compare, sequence, or manipulate.
1. Screening wild-type and mutant samples
PCR is widely used to distinguish wild-type and mutant genotypes.
For example, if a mutation introduces an insertion or deletion, primers can be designed so that the wild-type allele produces a 500 bp PCR product while the mutant allele produces an 800 bp product.
After PCR, agarose gel electrophoresis can then distinguish the two genotypes by band size.
Wild type: 500 bp
Mutant: 800 bp
This type of PCR-based screening is commonly used for insertions, deletions, knockouts, knock-ins, and other engineered or naturally occurring sequence differences.
2. Detecting SNPs
PCR can also be used to detect single nucleotide polymorphisms, or SNPs.
A single nucleotide difference usually cannot be distinguished directly as a 1 bp size difference on a standard agarose gel. Instead, PCR can convert the sequence difference into a difference in amplification.
In allele-specific PCR, for example, primers are designed so that amplification depends on whether the nucleotide at the SNP position matches the primer sequence. A matching primer can support efficient amplification, whereas a mismatch near the 3′ end can strongly reduce amplification.
The resulting genotype can therefore be inferred from the presence or absence of PCR products.
Other approaches include PCR-RFLP, probe-based genotyping, high-resolution melting analysis, and DNA sequencing of PCR products.
3. Preparing DNA for sequencing
PCR is frequently used to amplify a target region before DNA sequencing.
Instead of sequencing an entire genome or plasmid, researchers can selectively amplify the region of interest, confirm that a PCR product of the expected size has been obtained, purify the product, and then perform Sanger sequencing or another sequencing method.
This can be used to confirm mutations, SNPs, cloned inserts, or other sequence features.
4. Preparing DNA fragments for cloning
PCR products can also be used as DNA inserts for molecular cloning.
A gene or other target sequence can be amplified by PCR and introduced into a plasmid vector using restriction enzyme cloning, Gibson assembly, or other cloning strategies.
PCR is also frequently used after cloning to screen colonies and confirm whether the desired insert is present.
Why run PCR products on an agarose gel?
After PCR, it is not possible to determine simply by looking at the reaction tube whether the correct DNA fragment has been amplified.
Agarose gel electrophoresis separates DNA fragments according to their electrophoretic mobility. Because DNA has a negatively charged phosphate backbone, it migrates toward the positive electrode in an electric field.
The agarose matrix acts as a molecular sieve. Smaller DNA fragments generally migrate farther through the gel, whereas larger fragments migrate more slowly.
This makes it possible to estimate DNA fragment size by comparing an unknown band with DNA fragments of known size.
What is a DNA ladder?
A DNA ladder is a mixture of DNA fragments with known lengths.
For example, a ladder may contain fragments of 100 bp, 200 bp, 300 bp, 500 bp, 1000 bp, and other defined sizes.
By running the ladder alongside experimental samples, the position of an unknown band can be compared with known DNA fragment sizes.
However, DNA fragment size and migration distance do not have a simple linear relationship.
For example, if an unknown band appears exactly halfway between the 500 bp and 1000 bp ladder bands, it should not automatically be interpreted as 750 bp.
Why use the logarithm of DNA fragment size?
Over an appropriate size range, the logarithm of DNA fragment size is approximately related to migration distance in agarose gel electrophoresis.
This makes it possible to construct a semi-log calibration using DNA ladder bands.
For each ladder band, the migration distance is measured and the known fragment size is converted to log10(bp). A regression relationship can then be generated between migration distance and log10(fragment size).
The migration distance of an unknown band can be entered into this calibration to estimate its DNA fragment size.
This provides a more quantitative estimate than simply judging the band position visually.
How to estimate DNA fragment size from a gel image
BioChemCalc provides an Electrophoresis Band BP Calculator for estimating DNA fragment size directly from an agarose gel image.
A typical workflow is:
1. Load the gel electrophoresis image.
2. Select the positions of DNA ladder bands.
3. Enter the known fragment sizes of the ladder bands.
4. Select the position of the unknown sample band.
5. Generate a semi-log calibration.
6. Estimate the DNA fragment size of the sample band.
This allows the relationship between migration distance and log10(bp) to be evaluated directly from the gel image without manually measuring distances and performing regression calculations in a separate spreadsheet.
Compare the expected and observed PCR product sizes
For PCR experiments, the most important question is not simply whether a band is visible.
The key question is whether the observed band size agrees with the expected amplicon size predicted from primer design.
For example, if primer design predicts a 700 bp PCR product and a clear single band appears near 700 bp, this supports successful amplification of the intended target region.
In contrast, if a 700 bp product is expected but a strong band appears near 1200 bp, a different DNA region may have been amplified.
Multiple bands may indicate non-specific amplification, while strong low-molecular-weight bands can sometimes be associated with primer dimers or other small amplification products.
Band size alone does not prove the identity of a PCR product, so sequencing or another confirmatory method may still be required.
Agarose concentration also affects DNA separation
DNA mobility in agarose gel electrophoresis is influenced by more than fragment size alone.
Agarose concentration, applied voltage, DNA conformation, buffer composition, and other experimental conditions can affect migration.
In general, lower-percentage agarose gels are better suited for separating relatively large DNA fragments, while higher-percentage gels provide better separation of smaller fragments.
For accurate size estimation, the target DNA fragment should therefore fall within a well-resolved region of the gel and within the useful range of the selected DNA ladder.
From PCR primer design to DNA analysis
PCR primer design and gel electrophoresis may appear to be separate experimental steps, but they are parts of the same analytical workflow.
Select the DNA region of interest
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Design PCR primers
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Predict the expected amplicon size
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Amplify the target DNA by PCR
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Run agarose gel electrophoresis
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Estimate the observed DNA fragment size
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Compare expected and observed sizes
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Genotyping / SNP analysis / Sequencing / Cloning
BioChemCalc provides tools for both the beginning and the end of this workflow.
PCR Primer Design Tool
Select PCR primer candidates and evaluate melting temperature, GC content, hairpin formation, and primer dimerization.
Electrophoresis Band BP Calculator
Estimate DNA fragment size from an agarose gel electrophoresis image using DNA ladder-based semi-log calibration.
PCR is not simply a way to make more DNA. It is a way to selectively isolate a genetic question and convert it into something that can be observed, compared, sequenced, or manipulated.
And one of the first questions after PCR remains remarkably simple:
Did we amplify the DNA fragment of the expected size?
References
Lee PY, Costumbrado J, Hsu CY, Kim YH. Agarose Gel Electrophoresis for the Separation of DNA Fragments. Journal of Visualized Experiments. 2012;(62):3923. doi:10.3791/3923.
Stellwagen NC. Accurate molecular weight determinations of deoxyribonucleic acid restriction fragments on agarose gels. Biochemistry. 1983;22(26):6180-6185. doi:10.1021/bi00295a022.
Kim S, Misra A. SNP genotyping: technologies and biomedical applications. Annual Review of Biomedical Engineering. 2007;9:289-320. doi:10.1146/annurev.bioeng.9.060906.152037.