Spotlight Luminescence, UV & Image Analysis
Determination of DNA and RNA Melting Point on UV-VIS Photometer SPECORD®
PLUS Alexandra Kästner, Molecular Spectroscopy, Analytik Jena AG, 07745 Jena, Konrad-Zuse-Str. 1
Nucleic acids are biological molecules essential for life. Together with proteins nucleic acids make up the most important macro molecules where each is found in abundance in all living things. They allow organisms to transfer genetic information from one generation to the next. There are two types of nucleic acids: deoxyribonucleic acid, better known as DNA and ribonucleic acid, better known as RNA. Their names are derived from type of sugar, ribose, contained within the molecules. In living organisms, DNA does exist as a double helix structure. The stability is achieved by stacking and the hydrogen bonds between the bases attached to the two strands. The four bases found in DNA are adenine (abbreviated A), cytosine (C), guanine (G) and thymine (T). They form complementary pairs: The nucleotides hydrogen bond to another nucleotide base in a strand of DNA opposite to the original. This bonding is specific, and adenine always bonds to thymine (and vice versa) and guanine always bonds to cytosine (and vice versa).
Variations in pH or heating can affect to a structure modification of DNA, where the double-stranded deoxyribonucleic acid unwinds and separates into single-stranded strands through the breaking of hydrogen bonding between the bases. This denaturation process is called as DNA melting and leads to increasing of absorbance (hyperchromic effect).
From there one of the most commonly used and simplest techniques for the DNA melting point determination is spectroscopic determination by UV absorption. The absorption spectrum is recorded against the dependence of the temperature where the turning point of the graph describes the exact melting point. The temperature where at the half of the DNA exists as single strands, is called melting point (Tm).
Theory
All nucleic acids absorb strongly in the UV region due to the heterocyclic ring structure associated with each of the four bases. Typically absorption maximum is observed at a wavelength of around 260nm, although this is pH dependent. The versatility of DNA comes from this fact that the molecule is actually double- stranded. The bonding between cytosine with guanine is generally stronger than adenosine/thymine base-pairing. [1]
The amount of cytosine and guanine (called the ‘GC content’) can be estimated by measuring the temperature at which the DNA melts. Higher temperatures are associated with a high GC content. So by melting point determination it is possible to classify bacteria because the GC content in the DNA is an important fact of any organism. [2]
DNA denaturation can also be used to detect sequence differences between two nucleic acids of different origin. DNA is heated and denatured into single-stranded state, and the mixture is cooled to allow strands to rehybridise. Hybrid molecules are formed between similar sequences and any differences between those sequences will result in a disruption of the base-pairing [3].
In field of disease research rare gene mutations can be detected because mutated DNA sequences melt at lower temperatures than ‘normal’ ranges. Furthermore the process of DNA melting plays an important role in molecular biology techniques, notably in the polymerase chain reaction (PCR).
Experimental
DNA melting point determination is performed on SPECORD® of the peltier temperatured cell holder. SPECORD®
200 PLUS by means 200 PLUS is a real double beam
photometer for the wavelength range of 190-1100nm. It has a fixed spectral bandwidth of 1.4nm and two photodiodes. [4]
The peltier temperatured accessory enables applications that demand high temperature accuracies like accurate protein analyses and examination of photochemical reactions or like this application the determination of DNA melting point. Temperature control of the cell holder is performed via a separate control unit and can be regulated in a temperature range of -5 to 105°C and with an accuracy of ± 0.1°C (Figure 1). The controlling sensor used is a measuring sensor located at the outer bottom corner of the cell block. In addition to the controlling sensor, the cell holder contains two further sensors for optional monitoring of either the holder or the cell temperature. The cell sensor is specially designed for ultra-micro cells with round PTFE stopper. It may remain in the cell during the analytical measurement (Figure 2). [5]
Figure 2. Cell sensor for ultra- micro cells Sample material
Two different DNA samples were analysed for DNA melting point determination. The first sample was Plasmid DNA from bacteriophage Lambda. Plasmid is a circular double strand DNA molecule that is not integrated into bacterial chromosome.
The second sample was an animal DNA that is from a thymus of a calf.
Lambda DNA stock solution was diluted with water for the determination of plasmid DNA. The optimum dilution is achieved when the absorption maximum lies between 0.1 and 1 Abs. The spectrums of the samples are taken for this purpose between 220nm and 300nm and the peak maximum has been found (Figure 3). The analysis of a 100µL DNA solution was performed on an ultra-micro cell against molecular water used as a blank solution. The final melting point measurement was carried out in ‘simultaneous’ mode between the temperature range of 25°C to 70°C and at 260nm, where the spectrum of DNA has a maximum at this wavelength. The heating of the sample was carried out with an increase of 1°C per minute where a measuring data was recorded every 1°C.
The sample preparation and recording of the absorbance spectrum of the calf thymus DNA was performing analogously to the Plasmid- DNA. The melting point determination was carried out in the ‘cyclic‘ measurement mode with a start temperature of 70°C and a end temperature of 95°C. Whereby in the range from 70°C to 80°C the optical measurement was performed every 0.2°C; in range from 80°C to 90°C every 0.1°C and from 90°C to 95°C again every 0.2°C. The temperature was controlled with an accuracy of 0.1°C. During the entire analysis the exact temperature was monitored by means of the measuring sensor in the cell.
Results and discussion
The melting process of the DNA-samples could be shown very well in both measurement modes (Figure 4). Because of the base stacking the structure modification of the DNA is performed within a narrow temperature interval. At a defined temperature the double helical structure will be broken down very fast where the absorbance will increase rapidly. In ‘cyclic‘ measurement mode it is possible to set different intervals of several temperature ranges. The melting curve of the thymus DNA could be shown more accurately by setting smaller measurement points in the range where absorbance increases (Figure 6).
Figure 1. (a,b,c) SPECORD® PLUS with peltier temperated cell holder
The melting points of both DNA samples are derived from the peak maximum of first deviation of the melting curves. The absorbance increase of the bacterial DNA has taken at lower temperatures compared to the thymus DNA. The melting temperature of plasmid DNA and calf thymus DNA are determined at 46.3°C (Figure 5) and at 85.7°C (Figure 7), respectively.
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