Quantifying the building blocks of DNA is now easier thanks to a novel technique

Date:
June 24, 2020
Source:
University of Helsinki
Summary:
A highly sensitive and easy-to-use technique applicable for tissue
samples can be useful, for example, to researchers specialized in
mitochondrial diseases and cancer.



FULL STORY ==========================================================================
DNA, or deoxyribonucleic acid, carries genetic information in the
chromosomes of cell nuclei and in mitochondria. DNA is a double helix
composed of two long, coiled strands of polynucleotide chains. The
chains are composed of four different deoxyribonucleosides attached to
each other through phosphate bonds: deoxyadenosine (dA), deoxythymidine
(dT), deoxycytidine (dC) and deoxyguanosine (dG).


==========================================================================
When DNA is replicated during cell division or when it requires
repair, DNA polymerase enzymes produce a new strand of DNA, using deoxyribonucleoside triphosphates (dNTP) as its building blocks. The
energy needed for this synthesis comes from the chemical energy charged
in the phosphate bonds of the dNTPs.

With previously available techniques, measuring dNTP concentration has
been a challenge particularly in the case of samples with little DNA
synthesis and, thus, a low dNTP concentration. Now, researchers have
developed a technique which allows measurement of dNTP concentrations
with much improved sensitivity, for example, from small tissue samples collected from mice.

The study was published in the Nucleic Acids Research journal.

"Our assay is based on DNA polymerase and other materials commonly
used in molecular biology laboratories. The assay requires no special
equipment or radioactive substances," Docent Jukka Kallija"rvi says.

A sensitive method needed for investigating mitochondrial diseases
Mitochondria are organelles that are responsible for cellular respiration,
but they also have many other functions. The synthesis of pyrimidine nucleosides, such as thymidine and cytidine, is directly dependent on the mitochondrial respiratory chain. In mitochondrial diseases, deficiency in
the respiratory chain function can cause disturbances in the biosynthesis
of nucleosides and their phosphorylated counterparts nucleotides.



==========================================================================
In rapidly dividing cells, such as those in the bone marrow and cancerous tissue, dNTP concentrations are high, while in cells that divide slowly
or not at all, such as liver and muscle cells, the concentrations are
very low.

However, all cells need some dNTPs for repairing DNA damage and
maintaining mitochondrial DNA.

In patients suffering from a mitochondrial disease known as GRACILE
syndrome and in its mouse model, the main symptoms appear in the liver
and kidneys.

These tissues have few dividing cells and, therefore, also a low dNTP concentration.

"I started measuring the amount of dNTPs in mouse tissue and quickly
realized that the previous method was not suitable for tissue samples,"
says doctoral student Janne Purhonen.

A novel robust and sensitive assay solved the problem In the new dNTP
assay developed by the researchers, significant innovations included
the use of a DNA polymerase, which tolerates impurities in the sample,
as well as the utilisation of a sensitive fluorescent dye that separated double-stranded from single-stranded DNA.



==========================================================================
One of the technical challenges was the disturbance potentially caused
by ribonucleotides, the building blocks of RNA that may be thousands of
times more abundant than dNTPs in tissues.

Developing the technique, Purhonen realized that the addition of another
enzyme that cuts the DNA strand at an erroneously added ribonucleotide
allows differentiation of the correct and false reaction products based
on their melting temperature. This way, the signal due to erroneously incorporated ribonucleotides could be removed.

The study was carried out by the GRACILE research group headed by Docent
Jukka Kallija"rvi and Professor Emerita Vineta Fellman at the Folkha"lsan Research Center.

"We hope the assay will turn out useful to researchers of mitochondrial diseases as well as elsewhere in biological and medical research,"
Kallija"rvi sums up.

v

========================================================================== Story Source: Materials provided by University_of_Helsinki. Note:
Content may be edited for style and length.


========================================================================== Journal Reference:
1. Jukka Kallija"rvi, Vineta Fellman, Allison E McDonald, Rishi
Banerjee,
Janne Purhonen. A sensitive assay for dNTPs based on long
synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant
high-fidelity DNA polymerase. Nucleic Acids Research, 2020; DOI:
10.1093/nar/gkaa516 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/06/200624100029.htm

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