The DNA is an inheritance and storage unit of every organism on earth. By doing a DNA test we can identify any individual using the DNA fingerprinting methods.
The DNA fingerprinting method often called “DNA test” used for the criminal identification, maternal or paternal identification, identification of an unknown individual, for knowing the history of a person and for distinguishing two individuals or species.
It is not only used for human DNA testing but it is also accepted for the plant research studies, species/speciation and evolutionary studies.
And why not!
The DNA test is one of the most accurate and advanced methods available in recent days.
The testing method is so simple, we just have to send our biological sample to the expert and they create our DNA profile.
However, it is very difficult to identify some biological sample, whether it contains a DNA or not. Even if it carries a DNA, which type of DNA or whose DNA the sample contains, we don’t know.
Hence detecting DNA from an unknown biological sample is hard but not impossible.
In the present article, I will explain to you five methods using which you can detect the presence of DNA, you can also determine what type of DNA a sample has.
The methods used for the detection of DNA are:
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- UV- Vis Spectrophotometric analysis
- Fluorometric analysis
- DNA Precipitation
- DNA Gel electrophoresis
- Polymerase chain reaction
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I have sequentially enlisted 5 methods, you can use any one of this for detecting the presence of DNA However, every method has its own limitations which I will explain to you later.
Read our excellent article on DNA: DNA story: The structure and function of DNA
1. Uv-Vis Spectrophotometric method:
DNA absorbs UV light at 260nm wavelength.
By taking the 260/280 ratio, we can determine the presence of DNA in any biological sample.
The 260/280 ratio of pure DNA is nearly ~1.80 (1.77 to 1.88).
Why we have to do a spectrophotometric analysis first?
Because a sophisticated instrument like Nanodrop lite required only a single microliter of sample. Therefore if the sample quantity is too low (as in case of crime scene sample), we can confirm the presence of DNA by using only a minute amount of it.
If the ratio of 260/280 obtained between 1.77 to 1.88, DNA may present in our sample.
Note: if the sample is very low, after doing the spectrometric analysis use the sample directly for the DNA testing.
Read our article on A Comparative Review Between Qubit vs Nanodrop
2. Fluorometric analysis:
The fluorometric analysis is another method used for the detection of DNA, however, it is not as accurate as the 260/280 ratio.
In the Fluorometric analysis, the fluorescent dye is used to intercalate the DNA.
Once the Fluorochrome binds to the dsDNA, it emits the fluorescence which is detected by the detector.
Here, the fluorometric analysis provides one additional advantage over the spectroscopic analysis.
The fluorescent dye never binds to the single-stranded DNA, it intercalates between the hydrogen bonds of the dsDNA, hence it only detects the presence of dsDNA.
The accurate amount of dsDNA in any biological sample is determined by fluorometric analysis.
The disadvantage of the present method is that it can not determine the purity of DNA accurately.
Therefore we can not surely say that whether the calculated sample is DNA or not.
3. DNA precipitation:
DNA precipitation method is a simple and rapid method for detecting DNA in any biological sample.
Add chilled ethanol in a double volume of sample with a 1/10 volume of sodium acetate with it.
If the DNA is present into the sample it will precipitate in alcohol.
Although the method has a limitation too.
Sometimes a smaller amount of DNA can not be precipitated. For examples the DNA of amniotic fluid or hairshaft because the amount of DNA is very low and the precipitate can not form.
In that case, our sample might be wasted, once we add the alcohol and precipitate is not formed, the sample can not be processed further.
Read our articles on DNA precipitation:
4. Gel electrophoresis:
Agarose gel electrophoresis can also be used for detecting DNA from the unknown sample.
Mix the sample with the gel loading dye and run on the agarose gel, use 0.8% agarose gel because if the sample contains DNA, that DNA might be a genomic DNA.
Observe the results under UV transilluminator.
Here the EtBr used in the agarose gel preparation intercalates between the dsDNA if the DNA is present.
However, higher expertise required to detect the presence of DNA through agarose gel electrophoresis.
Differentiating between DNA and RNA is a hard task in agarose gel electrophoresis.
Read our article on Agarose gel electrophoresis
5. Polymerase chains reaction:
In the amplification reaction, using a specific set of random primers, the presence of DNA can be detected employing the polymerase chain reaction, however, the above-listed methods are more reliable than the PCR.
If amplification is not observed, you can not identify the reason whether it is because of the PCR failure or the DNA is absent.
Read an article on A Complete Guide of the Polymerase Chain Reaction
Use the Uv-Vis spectrophotometric analysis first for determining the presence of DNA.
Sample requirement for each method:
| Method | Sample requirement |
| Spectrophotometric analysis | 1-2µl |
| Fluorometric analysis | 8-10µl |
| DNA precipitation | 500-1000µl |
| Gel electrophoresis | 7-10µl |
| PCR | 3µl (~100ng/µl) |
Now I will explain to you how you can identify which DNA is present in the sample.
Once you confirm that the unknown sample is DNA, still you didn’t get your answer, what is the DNA sample is?
Suppose for instance if you receive a biological sample claimed a DNA, then start with here.
First, do the spectrophotometric analysis.
Step 1:
Doing UV visible spectrophotometry will give us a rough idea about the presence of DNA and the purity of DNA.
For instance the 260/280 ratio of our unknown sample ~1.90
In this case, the sample might contain the DNA but it is not a pure one.
Step 2:
In the next step do the Fluorometric analysis.
By doing this, we will get the exact concentration of the DNA present in the unknown sample.
For let say we get 260ng/µl.
Now we have a good quantity of DNA but it is not pure so we have to purify it.
Step 3:
In the next step precipitate the DNA using the alcohol as explained in the above section.
Once the precipitate observes wash the sample with 70% alcohol twice or thrice.
Check the 260/280 ratio.
Note: you can also use the ready to use DNA purification kit.
Here agarose gel electrophoresis is not needed. So don’t waste sample by doing it, skit it and go the PCR step.
Step 4:
Species-specific different random primer sets are now available commercially.
Suppose your unknown sample is an environmental sample, then at first, it might contain some bacterial DNA or animal DNA or human DNA.
Choose the primers with the best of your knowledge of sample collection and do amplification using standard procedure.
Step 5:
After amplification, run the sample on the agarose gel and analyze the amplification pattern.
See carefully which random primers are amplifying the DNA and note the data.
Step 6:
Send the sample for DNA sequencing.
After getting the DNA sequence, the lab representative will compare the DNA sequence with the sequences of different organisms and species and will find the exact match of our DNA.
If we perform all the steps perfectly, the results will surely 100% true.
You can also cross-check the results.
For instance, the DNA sequencing data revealed that the sample is of some kind of E.coli bacteria, you can amplify again the DNA using the E.coli species-specific primers.
If it amplifies, you can confirm the results and can send a report to the client very confidently.
Conclusion:
As in my point of view, Spectrophotometric analysis and DNA precipitation are two best methods for recognising DNA from the unknown sample because less sample is used to do so.
