DNA Preparation for sequencing

1. Preparing “DNA” for
Sequencing
Dr. Pavan K. J
M.Sc., Ph.D.,
Assistant Professor
Department of Biotechnology
G. M Institute of Technology
Davanagere, Karnataka,
India.

2. “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.”

3.  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 specialized, 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

4. 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.

5. 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.
No amplification:
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

6. Poor amplification:
Even though amplification occurs in some case, due to the
poor quality template, amplification and sequencing fail.
Background noise:
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.
The background noise makes the final sequencing results
non-conclusive.

7. 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.

8. DNA extraction is the first step in any genetics and
genomics technique. Well performed DNA extraction always
makes assay successful.
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.

9. 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.

10. 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.

11.  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.

12. 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.

13. 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).
https://www.youtube.com/watch?v=2rMvU6AzfZA
Nanodrop

14. 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
PCR fragments:
• 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

15.  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.

16.  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.

17.  Unused primers and dNTPs must be removed before
performing sequencing with the PCR products.
 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.

18. 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.

19. 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.

21. Note: before using column-based PCR product purification,
check the fragment capacity of the column.
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. with experience, dye-containing or
coloured PCR master mix is not recommended for
amplification as the dye may hinder or affect the sequencing
process.

22. 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

23. Summary:
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