“Preparing ‘DNA for sequencing’ is a crucial and important step in the DNA sequencing process. The quality and quantity of the template DNA are two key factors for getting better sequencing results.”
DNA sequencing is a process in which a sequence or the order of nucleotides can be determined.
DNA is a polynucleotide chain made up of sugar, phosphate and nitrogenous bases. In order to find out any alteration in the DNA sequence of the nucleotide chain of a gene, we have to examine its sequence.
A specialised, sophisticated, genetic technique or machine performs this function known as DNA sequencer and the process referred to as DNA sequencing. The major benefit of the present genetic technique is that it has the potential to discover new variations in a DNA sequence.
The technique becomes more aggressive, it any reference sequence it available.
In the present article, I will explain to you the process of how to prepare DNA for various sequencing platforms and what is the importance of doing it. But before going into the article let us quickly understand about DNA sequencing.
DNA sequencing and Steps:
The order of DNA nucleotides is read during DNA sequencing. Here based on the various platform the principle of sequencing vary. But in almost every platform, each single nucleotide read is recorded during the single strand amplification process.
DNA extraction, template preparation, library construction, sequencing, data gathering and results interpretation are some of the key steps in DNA sequencing. Extensive computational analysis is required to interpret the results.
Read two of our best article on DNA sequencing:
- What is DNA sequencing? A beginner’s guide
- DNA sequencing: history, definition, process, application and limitations.
Majorities of the DNA sequencing platforms work on the fluorescence chemistry, either fluorescent probe or dNTPs.
Common sequencing problems:
During sequencing, various types of conclusive and non-conclusive data are generated. Among them, some of the common problems you can encounter, are enlisted here.
poor quality and quantity template can’t be amplified and sequenced as various contaminant hinders in the reaction. No data are obtained in case of “no amplification”.
Even though amplification occurs in some case, due to the poor quality template, amplification and sequencing fail. Here, some portion of the entire template DNA is sequenced while some are not. Overall, the results of “poor amplification” are considered as non-conclusive.
Background or non-specific amplification signals are known as background noise appear as smaller peaks in between the original amplification peak or beneath the peak.
Not well-designed primers and template preparation are two key reasons for that. Here, with the template other DNA regions are also sequenced and amplified. Conclusively, the background noise makes the final sequencing results non-conclusive.
Weak base signals:
When it gets harder for Taq DNA polymerase to amplify the template, reading becomes even harder, this generates weak base signals. These data are not reliable for interpreting results.
All these problems occur due to inappropriate, contaminated and unpurified template DNA. In the next section, we will discuss some of the practical aspects of how to encounter these problems.
DNA for Sequencing:
The quality and the quantity of the template DNA has an important role in DNA sequencing. A good quality template performs well during sequencing without any background noisy sounds, gaps or non-amplifications. Thus it is crucial to quantify the template DNA before DNA sequencing.
The overall accuracy and specificity of the results depend on the template we have prepared.
The template DNA is a nucleotide sequence of plasmid, bacterial or other DNA which we wish to study.
DNA extraction is the first step in any genetics and genomics technique. Well performed DNA extraction always makes assay successful. Here for sequencing, not all the DNA extraction protocols perform well.
For salting-out method or salt-based DNA extraction, removing traces of salts and other ions are important otherwise its hurdle in the sequencing.
Isopropanol precipitation is a good option for both genomic and plasmid DNA precipitation. Washing plasmid DNA with 70% alcohol removes traces of all other contaminants such as salt, phenol or chloroform. However, before dissolving DNA, drying DNA is crucial to remove alcohol traces.
Also, use RNase or RNase containing kits for DNA extraction or plasmid isolation. RNA must be removed from the DNA sample.
If you are using the ready to use DNA extraction kit, don’t think that those are 100% reliable and accurate. The portion of silica, other reagents and salt can increase reaction failure chances.
Before eluting sample DNA or plasmid DNA, air dries the column containing the DNA. For increasing the quality, centrifuge the empty column to make the plasmid DNA dry properly. Elute DNA afterwards.
Notably, TE (tris-EDTA) buffer eluted DNA can’t work in DNA sequencing. The EDTA obstacle in sequencing. Always dilute or elute the plasmid or genomic DNA sample in nuclease-free water (especially in case of DNA sequencing). If you are using a CsCl (Cesium chloride) preparation, Cs or Cl ions should properly be removed before sequencing.
Now if you extracted DNA well (you get good precipitate), in the next step, DNA quantification and quality check must be performed prior to sequencing.
Check DNA quality spectrophotometrically, micro-spectrophotometer such as Nanodrop light is widely applicable in assessing the quality of the DNA. At the optical density of 260 nm, DNA absorbs the UV light. By taking the ratio of 260/280 nm, the purity of DNA can be determined.
The ratio of 260/280 nearly ~1.80 is considered as pure, however, DNA with 1.78 to 1.99 can be used as well. Usually, the DNA below the OD of 1.7 and above 2.0 are contaminated with either phenol, RNA or proteins.
Yet another key factor considered in assessing the quality of the template is the 230/260 ratio. The 230/260 value must greater than 1.0.
For DNA sequencing, the fluorometric analysis is generally avoided. The fluorescent quantification isn’t powerful enough unlike the spectroscopic analysis.
We had written a whole article on the comparison between qubit (a fluorochrome) vs Nanodrop light (spectrophotometer). Read it here: A Comparative Review Between Qubit vs Nanodrop.
Another factor for choosing DNA for the sequence is the quantity of DNA. Fewer DNA results in assay failure while more than sufficient amount of DNA cause non-specific amplification and background noise.
100 to 200ng DNA are generally sufficient for DNA sequencing. Now in the next step, as per the requirement of the assay, the DNA sample is diluted.
Quantity of various DNA samples:
- Ds DNA- 100 to 300ng
- Ss DNA- 20 to 50ng
- 100bp to 500bp- 10ng to 20ng
- 500bp to 2000bp- 20ng to 100ng
BAC plasmid- 500 to 100ng
In addition to this, the conventional gel electrophoresis method is also used to estimate the purity of DNA.
In this technique, our sample DNA run along with the standard one. If smear observed above or below the DNA bands, the sample falls under the contaminated category. Our sample DNA band must match with the size of a standard DNA sample.
Now, these methods are used for the genomic DNA and plasmid DNA but for the PCR product, the entire process is different.
Usually, the gene of interest amplified before DNA sequencing thus only the region we wish to study is amplified in the PCR, although, purification of PCR product is necessary.
The PCR amplicons or PCR products are the purest forms of DNA fragments. If we take OD of it, it can be nearly 1.80. Interesting these fragments are not pure actually.
Unused primers, dNTPs other chemicals blocks sequencing reaction. Especially the unused primers. Therefore we should remove it first.
Note: contaminant such as salt, chemicals, dNTPs, primers, phenol, protein and RNA have negative impact sequencing governed by Taq DNA polymerase.
Unused primers and dNTPs must be removed before performing sequencing with the PCR products. Here, is one catch!
The automated, semi-automated or traditional Sanger sequencing method works on the mechanism of chain termination through ddNTPs. Thus there is an appropriate ratio of dNTPs and ddNTPs in the reaction. Excess dNTPs unbalance this ratio.
Conclusively, the chain termination reaction can not perform well. Likewise, the set of PCR primers (remains in the PCR products) cause non-specific amplification.
The sequencing performed through the single amplification reaction. Unlike, the PCR amplification here, only a single primer is used. Thus the pair of PCR primers act as the sequencing primers and amplify two different templates. Overall, it imbalance the reaction and produce some non-conclusive results.
In addition to this, other ingredients like MgCl2, KCl, DMSO and albumin like buffer reagents increase or decrease the activity of Taq DNA polymerase during sequencing reaction.
Alcohol purification is usually not recommended for PCR product purification. Hereby using ready to use DNA purification kits, high yield and purified PCR products obtained.
One more quality check performed before sending the sample to sequencing that is gel run.
Run your template PCR fragments on 2.0 to 2.5% agarose gel. Getting more bands, non-specific or smear bands and primer-dimer bands indicates sample rejection for sequencing.
The PCR products are selected only when one single prominent DNA band of our interest obtained.
Note: before using column-based PCR product purification, check the fragment capacity of the column.
Let me explain to you what is it exactly!
Suppose if your PCR product length is 2000bp and your column elution capacity is 1000bp. In this case, all the fragments up to 1000bp are eluted in the final steps but only your template fragment of 2000bp could not be eluted. So the column is of no use.
PCR master mix used for PCR amplification also influences the sequencing process. As per my personal experience, dye-containing or coloured PCR master mix is not recommended for amplification as the dye may hinder or affect the sequencing process.
The DNA for sequencing is yet not ready. We have to perform DNA fragmentation and library preparation.
For extensive sequencing platforms like next-generation, whole-genome and pyrosequencing, fragmenting DNA is crucial. Larger DNA fragments aren’t precisely sequenced. Thus for generating longer reads or fore the entire genome, we first have to create various fragments.
Using restriction digestion or PCR based amplification, DNA fragmentation is done. After that, an oligonucleotide sequence of adapters ligated to every fragmented DNA. It creates the entire library or collection of various fragments. Individual fragments are then sequenced in the machine. Gene mapping and chromosome map information helps to arrange each fragment.
What to do?
- Purify DNA or PCR product before sequencing
- Check the quality and quantity
- 260/280 ratio of DNA between 1.77 to 1.9 should use in sequencing
- 100ng to 100microgram DNA is required
- Remove primer dimers, unused primers, dNTPs and other buffer reagents from the final PCR product
- Perform electrophoresis of DNA or PCR product for validating results
- The plasmid DNA must be free of other genome DNA or RNA.
- Genome DNA must not have RNA in it.
- Use ‘ready to use’ DNA purification kit
What not to do?
- Don’t use unpurified DNA
- Poorly amplified PCR products are not recommended
- Avoid unpurified PCR products
- Do not use a coloured master mix
Roadmap to success:
- Isolate good quality DNA
- Quantify DNA
- Select good quality and quantity of DNA
- Purify the PCR product
- Run-on agarose gel
- Remove all the impurities from the template
Conclusively, we can say, a high purified DNA sample is a key to success in DNA sequencing. Preparing DNA for sequencing is very crucial to get success. DNA sequencing is a costlier procedure, a single reaction failure cost you more than a thousand bugs. Therefore every step should be performed precisely.
Yang Y, Hebron HR, Hang J. A method for preparing DNA sequencing templates using a DNA-binding microplate. J Biomol Tech. 2009;20(3):165–171.