“A pure & good quality DNA has a 260/280 ratio between 1.7 to 1.8 and a yield between 100ng/µL to 300ng/µL is used for PCR, DNA sequencing and other downstream applications.”
DNA extraction results are only said to be good if the purity and yield of DNA are sufficient to perform a DNA test. Plenty of DNA extraction or isolation methods exist from manual to automated.
Though routine day genetic labs highly rely on spin-column DNA extraction or magnetic bead-based technology, manual methods are giving good results, if performed well.
Small lab sets or genetic centers can’t afford an automated DNA extraction system or even spin-column kits and therefore they have to depend on manual methods and assure their quality timely.
A salting-out method, phenol-chloroform method and enzyme-based DNA extraction methods are common to use. We here are talking about the commercial genetic testing centers and therefore not talking about plant DNA extraction.
For manual DNA isolation, purity and quantity is the biggest concern for researchers. On the contrary, magnetic bead technology or spin-column DNA extraction kits directly gives good quality DNA.
However, as we said, there are many factors why not all are using these techniques frequently. Put simply, to achieve a successful DNA extraction, the final product must be highly pure and sufficient enough to process further.
Here is the article focusing on how you can improve the purity and yield of DNA. But first, let us understand how to assess the purity and yield of DNA.
Assessing the purity and yield of DNA:
Four different techniques can be used to determine the purity and yield of DNA: physical examination, spectroscopic analysis, fluorometric analysis and agarose gel electrophoresis.
Over a period of time, after getting tons of experience in hands-on DNA extraction, you may evaluate or assess DNA purity and yield by visualizing the elutes or pellets.
When the DNA precipitates (whitish pellets appear), like a cotton thread, and is visible clearly, it may have good purity and quantity definitely. However, the instrumental analysis must be required to assess the quality before proceeding further.
When you elute DNA in the TE buffer, if you get a clear, well-dissolved DNA sample, without any precipitate left, your DNA’s quality can be good. But if it can’t be dissolved, is hard to dissolve or some precipitates still remain, it must not be a good quality elute or nucleic acid.
Spectroscopic analysis using Nanodrop light or any other spectroscope is the best technique to assess the DNA in terms of quality (purity) as well as quantity (yield).
This means that this technique is powerful enough to measure both parameters precisely. Briefly, the present technique relies on the absorbance spectra, when the light of two different wavelengths of 260 and 280 passes through the DNA, it measures the DNA and assesses its quality.
The present technique requires less sample input for analysis and it is faster as well.
The fluorometric analysis is sometimes also used especially to measure the yield of DNA. Fluorochromes bind to DNA molecules and emit fluorescence that is detectable.
A detector detects it and measures the amount of DNA present in a sample. The fluorometric analysis is less efficient as it takes more time to give results.
There are significant differences between both techniques, you can read this article to understand: A Comparative Review Between Qubit vs Nanodrop.
One of the most traditional, conventional and under-rated DNA assessment techniques is agarose gel electrophoresis. We will not explain the whole technique, you can read the article on it here: what is agarose gel electrophoresis.
Put simply, in an appropriate gel matrix only nucleic acid can pass. All other debris remains in the gel and pure DNA can be isolated after completion of the run.
The intensity of DNA bands, the run time and banding patterns tell a lot about the purity and quantity of DNA, even though it isn’t used for DNA purification.
A good quality, pure DNA bands are well-separate, having moderate intensity, clear and well-distinguished. However, to assess the results of a genomic DNA gel, you must have experience to analyze and interpret gel electrophoresis results.
DNA purity and quantity assessment help us to know whether our DNA is useful or not! If not, meaning, the purity or quantity might not be good. Assessment helps us to rethink whether to go for repeat extraction or optimize the DNA sample.
Usually, by using some physical, chemical or enzymatic treatment the issue can be resolved, here are some tips to increase the purity and quantity of DNA.
How to increase DNA purity and yield?
Here are some of the optimization options to increase the purity and yield of DNA.
Select a good method to isolate DNA:
Which method you choose to isolate your DNA, matters a lot, I strongly recommend using the PCI (Phenol-chloroform-isoamyl alcohol) method as it will give you more DNA than any other method. But you have to be careful while performing extraction, as purity is an issue with it.
To achieve purity along with high yield using the phenol-chloroform method use 3 step alcohol washing using ethanol.
Preparing the lysate:
The first step in DNA isolation is preparing the lysate which is a crude like-mixture of cells and cell debris. The lysate is obtained by disrupting cell walls or cell membranes. Common methods to do so are physical, chemical and enzymatic.
Among all three, the chemical method is though more reliable and trusted, physical methods using resigns or magnetic beads are more accurate too. Chemicals such as SDS or triton X100 are often used in preparing lysate which digests the protein portion of a cell.
The use of enzymes such as proteinase K, peptidase, or pepsins is also a good option to prepare a lysate. I personally use a combination of chemical and enzymatic methods to prepare a good quality lysate.
It is important, because a good, well-separated and clear lysate is the first step in achieving the purity of DNA. In addition, when the sample is lysed correctly, the yield also increases.
In a well-lysed sample, all the cell debris settles at the bottom of the centrifuge tube after spinning, meaning, the supernatant will be well-separable. So use a good combination of enzyme-chemical to prepare lysate.
Semi-automated and automated DNA isolation methods rely on physical techniques using magnetic beads and resins.
Spin the sample well:
Proper centrifugation is very important to achieve a good quality of DNA. lysate prepared in the first step must be well-separatable in order to isolate the nucleic acid.
If the sample isn’t centrifuged at the correct RPM, you may get bad DNA isolates in terms of purity and quantity. For instance, if lysates spin at lower RPM, the solid and liquid parts can’t be separated accurately.
Centrifugation is a technique for biomolecule separation using a spinning wheel. The high-speed spinning of biological samples such as cells is separated into two phases; solid and liquid. RPM- revolution per minute is the unit to measure centrifugal force.
Ideally, the lysate or cell lysate should be centrifuged at 3000 to 10,000 rpm for 5 to 15 minutes. Noteworthy, centrifuging at high speed also decreases the quality of the final product.
Washing DNA thoroughly:
DNA extraction completes in three steps; sample processing, sample washing and DNA dissolving. Washing is one of the important steps in achieving purity. Washing is defined as, repeating centrifugation of DNA precipitates using alcohol.
Repeat washing until clear DNA pellets are observed, although keep in mind not to wash more than 3 times as it decreases the yield. To wash DNA with alcohol, you can use ethanol, methanol or isopropanol.
We strongly recommended using ethanol (70%). If you want to learn more about how things work, you can read this article: DNA precipitation and precipitation protocol.
Silica-binding chemistry is used for DNA washing as well, and also a trustworthy technique for isolating pure DNA samples. Silica-gel or elution method is widely accepted across the world for purification of low-quality DNA and isolating DNA from minute amounts of samples.
A distinct-sized silica gel is filled in the centrifuge tube which only allows the nucleic acid to pass through it and not other cell debris. Even a minute amount of DNA, if present in a sample can be eluted out using this technique.
In comparison to the alcohol-based purification technique, the spin-column technique is costlier but is accurate too. Nearly all nucleic acid elutes obtained by the present technique have a 260/280 ratio of ~1.8 and a quantity of nearly 100 to 120ng/µL.
Incubating the sample:
Usually, for routine DNA extraction, especially from the blood we aren’t incubating samples in boiling water, because it isn’t needed. However, for a smaller amount of sample, dried blood spot or any other tiny sample, a small modification may help you to increase yield and purity.
After treating the sample with the lysis buffer, heating the sample at 65℃ for 1 hour or 95℃ for 15 minutes helps to disrupt the cell wall and digest protein effectively.
Note that if you are using the proteinase K method, first incubate the sample as per the manufacturer’s protocol (for proteinase K) and then incubate in a boiling water bath.
Cleaning samples using the spin column may give you pure and high yield DNA. This method is mostly employed for insect DNA isolation when the sample amount is too low.
The use of RNase:
How can we purify samples? By removing other contaminants such as proteins or RNA present in the sample. RNA is the common contaminant present with the DNA when using the manual method of DNA extraction.
To improve the quality of DNA, you can use the RNase enzyme. The RNase is a nuclease that cleaves RNA molecules and destroys them, along with proteinase K, RNase may help a lot in crucial DNA extraction experiments.
Noteworthy, refer to the manufacturer’s instructions to use RNase in the experiments. Use appropriate concentration as advised by the manufacturer.
Use of state of art technology:
Traditional methods for DNA extraction are only working for samples such as blood or tissue, although for samples such as saliva probably PCI-like methods can’t work effectively.
Meaning, though purity can be achieved; a good concentration of DNA can’t be obtained. For such samples, use a ready-to-use DNA extraction kit, spin-column kit or Chelex resin method. Chelex 100 resins work so effectively as it not only removes contaminants but also deactivate DNase that destroys DNA.
Saliva samples are commonly used in DNA testing and it is a non-invasive technique for testing therefore recommended by experts. But getting DNA from the saliva isn’t an easy task.
If you have high sample loads, you can use the magnetic bead method or automated DNA extraction technique which gives a high yield.
Factors that affect the purity and yield of DNA:
Many factors decide what we get after DNA extraction. The purity and quantity of the nucleic acid we get depend on the factors we listed here:
- The experience and expertise of the researcher
- Quality of chemicals and solutions used
- Validation of instruments used
- Use of starting material
- Sample type used for DNA extraction
Why is DNA purity and yield important?
Playing with DNA isn’t an easy task as there are many possibilities of false-positive or false-negative results, once you will trap in problems of ambiguous results, you can’t resolve them.
And therefore purity and yield matter a lot in every downstream application. Let’s understand it with an example, Suppose our sample is contaminated with a bacterial sample. We wish to amplify a 200bp fragment of DNA to know whether it is present or not and a homologous bacterial gene gets amplified instead.
That’s a false-positive result. And we don’t want that, right! It will create more problems if the sample is of the patient. Similarly, phenol is a compound that prevents DNA amplification during DNA sequencing or PCR and therefore it must be removed prior to use. We can say purity is a big factor in genetic experiments.
The concentration of DNA:
“DNA concentration is the amount of DNA required to perform a specific experiment.” Note that, the concentration isn’t the quantity of DNA. A 10µL sample may have either 100ng/µL or 250ng/µL concentration.
The concentration of DNA depends on how much quantity of TE buffer or water you are adding to dissolve the DNA.
Suppose you dissolve a low yield DNA in 100 µL of TE buffer and a high concentration of DNA in the same amount of TE buffer the concentration of both varies.
Here is the list of samples we have extracted, dissolved in 100 µL of TE buffer using the PCI method.
|Sample number||TE buffer added (quantity)||Purity (260/280 ratio)||Concentration|
So we have 10 samples with the quantity of 100 µL but each has a different concentration. So we need to dissolve the sample further in order to reach the concentration required for the assay.
For example, we need 30 to 50ng DNA in PCR, so we need to dissolve every sample further. Similarly, we need 100ng DNA for restriction digestion we have to alter the concentration using the TE buffer. Keep in mind that purity also matters, as we explained above.
Effect of low quality DNA:
Now the question arises in mind: what are the consequences of low-quality DNA! Low quality DNA meaning that isn’t appropriate for performing some crucial experiments though it has DNA.
Contaminants present in a sample may stop or influence the reaction negatively. Major players are protein and other chemical traces. For instance, it is very hard to amplify the DNA with 1.22 (260/280 ratio) purity of DNA, Because it is impure!
Low-quality DNA meaning, it has a higher or lower 260/280 ratio than ~1.80, lower or too high concentration, unclear elute, undissolved DNA and contaminant DNA.
The quality and quantity of DNA depend on many factors which we already have listed above. The method for extraction, use of proteinase K and RNase, use of alcohol and use of state-of-art technology will definitely help you to improve the quality of DNA.
Technically, though, a range between ~1.77 and ~1.88 is accepted for routine use. But I highly recommended using a ready-to-use DNA extraction kit or elution technique if you can afford it.